Multifunctional radical quenchers for the treatment of mitochondrial dysfunction

ABSTRACT

The present disclosure provides methods for identifying therapeutic agents that are multifunctional radical quenchers. It also provides compounds of formula: 
                         
and pharmaceutically acceptable salts thereof, compositions comprising these compounds, and methods of using these compounds in a variety of applications, such as treatment or suppression of diseases associated with decreased mitochondrial function resulting in diminished ATP production and/or oxidative stress and/or lipid peroxidation.

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No.14/009,437 filed Oct. 16, 2013, which is a national stage filing ofPCT/US2012/032108 filed Apr. 4, 2012, which claims priority to61/471,346 filed Apr. 4, 2011, all of which are incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

The present disclosure provides methods for identifying therapeuticagents that are multifunctional radical quenchers. It also providesbiologically active compounds multifunctional radical quenchers andpharmaceutically acceptable salts thereof, compositions comprising thesecompounds, and methods of using these compounds in a variety ofapplications, such as treatment or suppression of diseases associatedwith decreased mitochondrial function resulting in diminished ATPproduction and/or oxidative stress and/or lipid peroxidation.

SUMMARY OF THE INVENTION

Mitochondria are intracellular organelles responsible for a number ofmetabolic transformations and regulatory functions. They produce much ofthe ATP employed by eukaryotic cells. They are also the major source offree radicals and reactive oxygen species that cause oxidative stress.Consequently, mitochondrial defects are damaging, particularly to neuraland muscle tissues which have high energy level demands. Thus, energeticdefects have been implicated in forms of movement disorders,cardiomyopathy, myopathy, blindness, and deafness (DiMauro et al. (2001)Am. J. Med. Genet. 106, 18-26; Leonard et al. (2000) Lancet. 355,299-304). There are a number of mitochondrial diseases resulting fromboth nuclear and mitochondrial genetic defects, and the underlyingbiochemistries of these diseases tend to be rather similar. They includeincreased lactate production, diminished respiration and ATP production,and reflect the consequences of oxidative stress. This inventiondescribes novel compounds for the treatment or suppression of diseasesassociated with decreased mitochondrial function resulting in diminishedATP production and/or oxidative stress and/or lipid peroxidation. Theinvention also describes use of these compounds for the treatment ofmitochondrial disorders, including but not limited to Friedreich'sataxia, Leber's Hereditary Optic Neuropathy, Kearns-Sayre Syndrome,Mitochondrial Encephalomyopathy with Lactic Acidosis and Stroke-LikeEpisodes and more generally, any disease associated with impairment ofenergy production and mitochondrial function. Aging may also involvedecreased mitochondrial function and diminished ATP production, and thetherapeutic agents described here may also find utility in mitigatingthe effects of aging.

Thus, in one aspect, the disclosure provides a method for identifying atherapeutic agent, comprising assaying a test compound for the abilityto:

a) quench mitochondrially generated reactive oxygen species (ROS);

b) quench lipid radicals produced by mitochondrially generated ROS;

c) augment ATP production, and

d) afford cytoprotection to cells under oxidative stress,

-   wherein such a test compound is a candidate therapeutic agent for    treating or protecting mitochondria with respiratory chain lesions.

Another aspect of the disclosure provides compounds of formula (I)

and pharmaceutically acceptable salts thereof.

Another aspect of the disclosure provides compounds of formula (II)

and pharmaceutically acceptable salts thereof.

Another aspect of the disclosure provides compounds of formula (II-A)

and pharmaceutically acceptable salts thereof.

Another aspect of the disclosure provides pharmaceutical compositionscomprising the compounds and salts of the disclosure and an appropriatecarrier, excipient or diluent. The exact nature of the carrier,excipient or diluent will depend upon the desired use for thecomposition, and may range from being suitable for veterinary uses tobeing suitable for human use. The compositions may optionally includeone or more additional compounds suitable for a use.

Another aspect of the disclosure provides methods of treating orsuppressing diseases associated with decreased mitochondrial functionresulting in diminished ATP production and/or oxidative stress and/orlipid peroxidation, comprising administering an effective amount of thecompound and salts of the disclosure.

Another aspect of the disclosure provides a method of treating orsuppressing one or more of Friedreich's ataxia, Leber's Hereditary OpticNeuropathy, Kearns-Sayre Syndrome, Mitochondrial Encephalomyopathy withLactic Acidosis and Stroke-Like Episodes, or Leigh syndrome, comprisingadministering an effective amount of the compound and salts of thedisclosure.

Another aspect of the disclosure provides a method of treating orsuppressing one or more of obesity, atherosclerosis, amyotrophic lateralsclerosis, Parkinson's Disease, cancer, heart failure, myocardialinfarction (MI), Alzheimer's Disease, Huntington's Disease,schizophrenia, bipolar disorder, fragile X syndrome, chronic fatiguesyndrome, and Leigh syndrome, comprising administering an effectiveamount of the compound and salts of the disclosure.

DESCRIPTION OF DRAWINGS

The results set forth herein, and the properties and characteristics ofthe compounds provided by the disclosure, can be advantageouslyunderstood with regard to the drawings.

FIG. 1 shows effect of the compounds of the disclosure on lipidperoxidation induced by oxygen radicals generated from thermaldecomposition of 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) (10mM) in phospholipids liposomes in Tris-HCl buffer containingC₁₁-BODIPY^(581/591) (200 nM) at 40° C. The red fluorescence decay ofthe probe was monitored with time (λ_(ex)=570, λ_(em)=600 nm). Relativefluorescence units are normalized to 100% intensity. Identical resultswere obtained in replicate experiments.

FIG. 2 shows effect of the compounds of the disclosure on lipidperoxidation induced by oxygen radicals generated from thermaldecomposition of AAPH in phospholipid liposomes in phosphate buffer at40° C. A protection against lipid peroxidation was measured by theirability to preserve the fluorescence of C₁₁-BODIPY^(581/591) in presenceof 10 mM AAPH. Relative fluorescence units are normalized to 100%intensity.

FIG. 3 shows effect of methylene blue on cellular ATP levels. Shown isrepresentative assay of intracellular ATP level in FRDA lymphocyte cellstreated with the indicated concentration of methylene blue and thecompound of disclosure.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the disclosure provides a method for identifying atherapeutic agent, comprising assaying a test compound for the abilityto:

a) quench mitochondrially generated reactive oxygen species (ROS);

b) quench lipid radicals produced by mitochondrially generated ROS;

c) augment ATP production, and

d) afford cytoprotection to cells under oxidative stress,

-   wherein such a test compound is a candidate therapeutic agent for    treating or protecting mitochondria with respiratory chain lesions.

In one embodiment, the disclosure provides the method wherein assayingthe ability of the test compound to quench mitochondrially generated ROScomprises: a) pretreating test cells with the test compound; b) treatingthe test cells with an agent that depletes cellular glutathione; c)treating the test cells with a fluorescent stain; and d) measuringfluorescence from the test cells, wherein a decrease in fluorescencefrom the test cells compared to a control indicates that the testcompound quenches ROS. In one embodiment, the disclosure provides themethod wherein the agent that depletes cellular glutathione is diethylmaleate, (R,S)-3-Hydroxy-4-pentenoate, or N-ethylmaleimide. In anotherembodiment, the agent is diethyl maleate. In another embodiment, thedisclosure provides the method wherein flow cytometry is used to measurefluorescence.

In one embodiment, the disclosure provides the method comprisingidentifying a test compound that reduces ROS production by at least 50%compared to control.

In one embodiment, the disclosure provides the method comprisingidentifying a test compound that reduces ROS production catalytically.Catalytic reduction of ROS production is determined by demonstratingthat the quantitative reduction in ROS exceeds the amount of testcompound employed.

In one embodiment, the disclosure provides the method wherein the ROScomprises superoxide, peroxide, hydroxyl radical, singlet oxygen ornitrogen oxides. In one embodiment, ROS is superoxide.

In one embodiment, the disclosure provides the method wherein quenchinglipid radicals produced by mitochondrially generated ROS comprisesmeasuring lipid peroxidation in a model membrane system. In oneembodiment, the model membrane system comprises phosphotidylcholineliposomes, submitochondrial particles (SMP), mitochondrial membranefractions. In one embodiment, the model membrane system comprisesphosphotidylcholine liposomes.

In one embodiment, the disclosure provides the method wherein assayingthe ability of the test compound to quench lipid radicals produced bymitochondrially generated ROS comprises: a) pretreating unilamellarphospholipid vesicles containing the embedded fluorophore with the testcompound; b) treating the phospholipid vesicles with a free radicalgenerator; and c) measuring fluorescence, wherein maintenance offluorescence from the phospholipid vesicles compared to a controlindicates that the test compound quenches lipid radicals produced bymitochondrially generated ROS. In one embodiment, the embeddedfluorophore is C₁₁—BODIPY^(581/591). In one embodiment, the free radicalgenerator comprises 2,2′-azo-bis(2-amidinopropane),2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH), or2,2′-azobis(2,4-dimethyl-valeronitrile) (AMVN). In one embodiment, thefree radical generator is a combination of ferrous sulfate, ascorbicacid and hydrogen peroxide. In one embodiment, the free radicalgenerator is 2,2′-azo-bis(2-amidinopropane). In one embodiment,measuring fluorescence is performed over a period of at least 20minutes.

In one embodiment, the method comprises identifying a test compound thatreduces lipid peroxidation in a model by at least 50% compared to acontrol.

In one embodiment, the method comprises identifying a test compound thatreduces lipid peroxidation in a model system by donation of a hydrogenatom or its functional equivalent. This can be determined by alsotesting a closely related analog of the test compound, which isincapable of donating a hydrogen atom. The test compound, after donationof the hydrogen atom or its functional equivalent, is resonancestabilized. This can be determined by also testing a closely relatedanalog of the test compound, which is incapable of comparable resonancestabilization.

In one embodiment, the method comprises identifying a test compound thatreduces lipid peroxidation in a model system and participates inelectron transfer reactions within the mitochondrial respiratory chain.This can be determined by testing electron donation to complex III,cytochrome c, or complex IV, or by demonstrating enhanced ATPproduction.

In one embodiment, the disclosure provides the method wherein assayingthe ability of the test compound to augment ATP production comprises: a)treating coenzyme Q₁₀ (CoQ₁₀) deficient test cells with the testcompound; b) incubating the test cells with the test compound; and c)measuring ATP levels in the test cells after incubation, wherein anincrease in ATP levels for the test cells compared to a controlindicates that the test compound augments ATP production.

In one embodiment, the method wherein assaying the ability of the testcompound to augment ATP production comprises starving CoQ₁₀ deficienttest cells for glucose prior to treatment with the test compound tolimit non-mitochondrial ATP production.

In one embodiment, the method assaying the ability of the test compoundto augment ATP production comprises wherein incubation of the test cellsis over at least 48 hours.

In one embodiment, measuring ATP levels comprises treatment withreagents to support the luciferin-luciferase reaction, which producesluminescence.

In one embodiment, the disclosure provides the method comprisingidentifying a test compound that augments ATP production by at least 5%compared to control. In one embodiment, ATP production is augmented atleast 10% compared to control.

In one embodiment, the method assaying the ability of the test compoundto augment ATP production comprises identifying a test compound thataugments ATP production by using electrons that have escaped from amitochondrial electron transport chain. This can be determined bydemonstrating that ROS decreases as ATP increases.

In one embodiment, the disclosure provides the method wherein assayingthe test compound for the ability to protect the test cells fromoxidative stress. In one embodiment, the test cells are Friedreich'sataxia fibroblasts. In another embodiment, the test cells areFriedreich's ataxia lymphocytes or CEM leukemia cells. In yet anotherembodiment, the test cells are from a broad range of inheritedmitochondrial diseases, including Friedreich's ataxia, Leber'sHereditary Optic Neuropathy, Kearns-Sayre Syndrome, MitochondrialEncephalomyopathy with Lactic Acidosis and Stroke-Like Episodes, orLeigh syndrome. In another embodiment, the test cells are from diseasesthat have a significant mitochondrial component or diseases in which thetissues are known to be under stress, including, but not limited toobesity, atherosclerosis, Parkinson's Disease, cancer, heart failure,myocardial infarction (MI), Alzheimer's Disease, Huntington's Disease,schizophrenia, bipolar disorder, fragile X syndrome, and chronic fatiguesyndrome.

In one embodiment, the disclosure provides the method wherein assayingthe ability to protect the test cells from oxidative stress comprises:a) pretreating the test cells with the test compound; b) treating thetest cells with an inhibitor of glutathione biosynthesis, or a compoundthat otherwise reduces the availability of reduced glutathione; c)incubating the test cells; and d) measuring cell viability of the testcells, wherein an increase in number of viable test cells compared to acontrol indicates that the test compound protects the cells fromoxidative stress. In one embodiment, the inhibitor of glutathionebiosynthesis is L-buthionine (S,R)-sulfoximine. In another embodiment,the inhibitor of glutathione biosynthesis isN,N,N′,N′-tetramethyldiazene-1,2-dicarboxamide. In one embodiment,measuring mitochondrial membrane potential is used to assess theviability of the test cells.

In one embodiment, the disclosure provides the method wherein the testcompound is a candidate therapeutic agent for treating or protectingmitochondria with respiratory chain lesions in concentration of lessthan 10 μM. In another embodiment, the concentration is less than 1 μM.

Another aspect of the disclosure provides compounds of formula (I)

or a pharmaceutically acceptable salt thereof, wherein

-   R¹ is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, —OR⁵, —SR⁵, —NHR⁵,    or —N(R⁵)₂, each optionally substituted with one to four    substituents selected from halogen, —CN, —NO₂, —N₃, C₁-C₆ alkyl,    halo(C₁-C₆ alkyl), —OR⁶, —NR⁶ ₂, —CO₂R⁶, and —CONR⁶ ₂;    -   where each R⁵ independently is hydrogen, C₁-C₆ alkyl, or        halo(C₁-C₆ alkyl);    -   where each R⁶ independently is hydrogen or C₁-C₆ alkyl;-   R² is hydrogen, halogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,    —OR⁵, —SR⁵, —NHR⁵, or —N(R⁵)₂, each optionally substituted with one    to four substituents selected from halogen, —CN, —NO₂, —N₃, C₁-C₆    alkyl, halo(C₁-C₆ alkyl), —OR⁶, —NR⁶ ₂, —CO₂R⁶, and —CONR⁶ ₂;-   R³ is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, —OR⁵, —SR⁵,    —NHR⁵, —N(R⁵)₂, each optionally substituted with one to four    substituents selected from halogen, —CN, —NO₂, —N₃, C₁-C₆ alkyl,    halo(C₁-C₆ alkyl), —OR⁷, —NR⁷ ₂, —CO₂R⁷, —OC(O)R⁷, —CON(R⁷)₂,    —OC(O)N(R⁷)₂, —NHC(O)N(R⁷)₂, —NHC(NH)N(R⁷)₂, C₃-C₈ cycloalkyl,    C₃-C₈cycloalkenyl, aryl, heteroaryl, and heterocycle, wherein each    cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle are    optionally substituted with R⁸;    -   where each R⁷ independently is hydrogen, C₁-C₆ alkyl, halo(C₁-C₆        alkyl), C₃-C₈ cycloalkyl, aryl, heteroaryl, heterocycle,        aryl(C₁-C₆ alkyl), C₃-C₈ cycloalkyl(C₁-C₆ alkyl), aryl(C₁-C₆        alkyl), heteroaryl(C₁-C₆ alkyl), or heterocycle(C₁-C₆ alkyl),        wherein each cycloalkyl, aryl, heteroaryl, and heterocycle are        optionally substituted with R⁸;    -   where each R⁸ independently is halogen, —CN, —NO₂, —N₃, C₁-C₆        alkyl, halo(C₁-C₆ alkyl), C₁-C₆ alkoxy, amino, C₁-C₆alkylamino,        or diC₁-C₆alkylamino; and-   R⁴ is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, —OR⁵, —SR⁵,    —NHR⁵, —N(R⁵)₂, each optionally substituted with one to four    substituents selected from halogen, —CN, —NO₂, —N₃, C₁-C₆ alkyl,    halo(C₁-C₆ alkyl), —OR⁷, —NR⁷ ₂, —CO₂R⁷, —OC(O)R⁷, —CON(R⁷)₂,    —OC(O)N(R⁷)₂, —NHC(O)N(R⁷)₂, —NHC(NH)N(R⁷)₂, C₃-C₈ cycloalkyl, C₃-C₈    cycloalkenyl, aryl, heteroaryl, and heterocycle, wherein each    cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle are    optionally substituted with R⁸;-   provided at least one of R³ or R⁴ is optionally substituted C₄-C₂₀    alkyl, optionally substituted C₄-C₂₀ alkenyl, or optionally    substituted C₄-C₂₀ alkynyl.

In one embodiment, the disclosure provides compounds of formula (I),wherein

-   R¹ is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, —OR⁵, —SR⁵, —NHR⁵,    or —N(R⁵)₂, each optionally substituted with one to four    substituents selected from halogen, —CN, —NO₂, —N₃, C₁-C₆ alkyl,    halo(C₁-C₆ alkyl), —OR⁶, —NR⁶ ₂, —CO₂R⁶, and —CONR⁶ ₂;    -   where each R⁵ independently is hydrogen, C₁-C₆ alkyl, or        halo(C₁-C₆ alkyl);    -   where each R⁶ independently is hydrogen or C₁-C₆ alkyl;-   R² is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, —OR⁵, —SR⁵, —NHR⁵,    or —N(R⁵)₂, each optionally substituted with one to four    substituents selected from halogen, —CN, —NO₂, —N₃, C₁-C₆ alkyl,    halo(C₁-C₆ alkyl), —OR⁶, —NR⁶ ₂, —CO₂R⁶, and —CONR⁶ ₂;-   R³ is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, —OR⁵, —SR⁵,    —NHR⁵, —N(R⁵)₂, each optionally substituted with one to four    substituents selected from halogen, —CN, —NO₂, —N₃, C₁-C₆ alkyl,    halo(C₁-C₆ alkyl), —OR⁷, —NR⁷ ₂, —CO₂R⁷, —OC(O)R⁷, —CON(R⁷)₂,    —OC(O)N(R⁷)₂, —NHC(O)N(R⁷)₂, —NHC(NH)N(R⁷)₂, C₃-C₈ cycloalkyl, C₃-C₈    cycloalkenyl, aryl, heteroaryl, and heterocycle, wherein each    cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle are    optionally substituted with R⁸;    -   where each R⁷ independently is hydrogen, C₁-C₆ alkyl, halo(C₁-C₆        alkyl), C₃-C₈ cycloalkyl, aryl, heteroaryl, heterocycle,        aryl(C₁-C₆ alkyl), C₃-C₈ cycloalkyl(C₁-C₆ alkyl), aryl(C₁-C₆        alkyl), heteroaryl(C₁-C₆ alkyl), or heterocycle(C₁-C₆ alkyl),        wherein each cycloalkyl, aryl, heteroaryl, and heterocycle are        optionally substituted with R⁸;    -   where each R⁸ independently is halogen, —CN, —NO₂, —N₃, C₁-C₆        alkyl, halo(C₁-C₆ alkyl), C₁-C₆ alkoxy, amino, C₁-C₆alkylamino,        or diC₁-C₆alkylamino; and-   R⁴ is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, —OR⁵, —SR⁵,    —NHR⁵, —N(R⁵)₂, each optionally substituted with one to four    substituents selected from halogen, —CN, —NO₂, —N₃, C₁-C₆ alkyl,    halo(C₁-C₆ alkyl), —OR⁷, —NR⁷ ₂, —CO₂R⁷, —OC(O)R⁷, —CON(R⁷)₂,    —OC(O)N(R⁷)₂, —NHC(O)N(R⁷)₂, —NHC(NH)N(R⁷)₂, C₃-C₈ cycloalkyl, C₃-C₈    cycloalkenyl, aryl, heteroaryl, and heterocycle, wherein each    cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle are    optionally substituted with R⁸;-   provided at least one of R³ or R⁴ is optionally substituted C₄-C₂₀    alkyl, optionally substituted C₄-C₂₀ alkenyl, or optionally    substituted C₄-C₂₀ alkynyl.

In one embodiment, the disclosure provides compounds of formula (I),wherein R¹ is C₁-C₆ alkyl. In another embodiment, R¹ is methyl.

In another embodiment, the disclosure provides compounds of formula (I),wherein R¹ is —OR⁵, —SR⁵, —NHR⁵, or —N(R⁵)₂.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (I), wherein R² is C₁-C₆ alkyl or C₁-C₆alkenyl. In another embodiment, the disclosure provides compounds asdescribed above with any reference to formula (I), wherein R² is C₁-C₆alkyl. In yet another embodiment, R² is methyl.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (I), wherein R² is C₁-C₆ alkenyl. Inanother embodiment, R² is vinyl or allyl. In yet another embodiment, R²is vinyl.

In another embodiment, the disclosure provides compounds as describedabove with any reference to formula (I), wherein R² is —OR⁵, —SR⁵,—NHR⁵, or —N(R⁵)₂.

In yet another embodiment, the disclosure provides compounds asdescribed above with any reference to formula (I), wherein R² is —OR⁵.In one embodiment, R² is —OR⁵ and R⁵ is hydrogen or C₁-C₆ alkyl. Inanother embodiment, R² is —OH. In yet another embodiment, R² is —O(C₁-C₆alkyl). In yet another embodiment, R² is —OCH₃.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (I), wherein R² is —NHR⁵, or —N(R⁵)₂. Inone embodiment, R² is —NHR⁵ and R⁵ is hydrogen or C₁-C₆ alkyl. Inanother embodiment, R² is —NH₂. In yet another embodiment, R² is—NH(C₁-C₆ alkyl). In yet another embodiment, R² is —NHCH₃. In anotherembodiment, R² is —N(C₁-C₆ alkyl)₂. In yet another embodiment, R² is—N(CH₃)₂.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (I), wherein R² is hydrogen or halogen.

In another embodiment, the disclosure provides compounds as describedabove with any reference to formula (I), wherein R² is hydrogen.

In yet another embodiment, the disclosure provides compounds asdescribed above with any reference to formula (I), wherein R² ishalogen. In one embodiment, R² is Br. In one embodiment, R² is Cl.

In one embodiment, the disclosure provides compounds of formula (I),wherein both R¹ and R² are C₁-C₆ alkyl. In another embodiment, both R¹and R² are methyl.

In another embodiment, the disclosure provides compounds of formula (I),wherein R¹ is C₁-C₆ alkyl and R² is C₁-C₆ alkenyl. In anotherembodiment, R¹ is methyl and R² is vinyl.

In another embodiment, the disclosure provides compounds of formula (I),wherein R¹ is C₁-C₆ alkyl and R² is —NHR⁵. In another embodiment, R¹ ismethyl and R² is —NH₂.

In another embodiment, the disclosure provides compounds of formula (I),wherein R¹ is C₁-C₆ alkyl and R² is —OR⁵. In another embodiment, R¹ ismethyl and R² is —OCH₃.

In another embodiment, the disclosure provides compounds of formula (I),wherein R¹ is C₁-C₆ alkyl and R² is hydrogen. In another embodiment, R¹is methyl and R² is hydrogen.

In another embodiment, the disclosure provides compounds of formula (I),wherein R¹ is C₁-C₆ alkyl and R² is halogen. In another embodiment, R¹is methyl and R² is Br or Cl.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (I), wherein R³ is optionally substitutedC₁-C₃ alkyl, optionally substituted C₁-C₃ alkenyl, or optionallysubstituted C₁-C₃ alkynyl. In one embodiment, R³ is optionallysubstituted C₁-C₃ alkyl. In another embodiment, R³ is methyl.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (I), wherein R³ is —OR⁵, —SR⁵, —NHR⁵, or—N(R⁵)₂.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (I), wherein R⁴ is optionally substitutedC₄-C₂₀ alkyl, optionally substituted C₄-C₂₀ alkenyl, or optionallysubstituted C₄-C₂₀ alkynyl. In another embodiment, R⁴ is optionallysubstituted C₅-C₂₀ alkyl.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (I), wherein R⁴ is optionally substitutedC₁-C₃ alkyl, optionally substituted C₁-C₃ alkenyl, or optionallysubstituted C₁-C₃ alkynyl. In one embodiment, R⁴ is optionallysubstituted C₁-C₃ alkyl. In another embodiment, R⁴ is methyl.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (I), wherein R⁴ is —OR⁵, —SR⁵, —NHR⁵, or—N(R⁵)₂. In this embodiment, R³ is optionally substituted C₄-C₂₀ alkyl,optionally substituted C₄-C₂₀ alkenyl, or optionally substituted C₄-C₂₀alkynyl. In another embodiment, R⁴ is optionally substituted C₅-C₂₀alkyl.

Another aspect of the disclosure provides compounds of formula (II)

wherein

-   X is halogen;-   R¹ is hydrogen, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, —OR⁷,    —SR⁷, —NHR⁷, or —N(R⁷)₂, each optionally substituted with one to    four substituents selected from halogen, —CN, —NO₂, C₁-C₆ alkyl,    halo(C₁-C₆ alkyl), —OR⁸, —NR⁸ ₂, —CO₂R⁸, —CONR⁸ ₂, C₃-C₈ cycloalkyl,    C₃-C₈ cycloalkenyl, aryl, heteroaryl, and heterocycle, wherein each    cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle are    optionally substituted with R⁹;    -   where each R⁷ independently is hydrogen, C₁-C₆ alkyl, or        halo(C₁-C₆ alkyl);    -   where each R⁸ independently is hydrogen, C₁-C₆ alkyl, halo(C₁-C₆        alkyl), C₃-C₈ cycloalkyl, aryl, heteroaryl, heterocycle,        aryl(C₁-C₆ alkyl), C₃-C₈ cycloalkyl(C₁-C₆ alkyl), aryl(C₁-C₆        alkyl), heteroaryl(C₁-C₆ alkyl), or heterocycle(C₁-C₆ alkyl),        wherein each cycloalkyl, aryl, heteroaryl, and heterocycle are        optionally substituted with R⁹;    -   where each R⁹ independently is halogen, —CN, —NO₂, —N₃, C₁-C₆        alkyl, halo(C₁-C₆ alkyl), C₁-C₆ alkoxy, amino, C₁-C₆alkylamino,        or diC₁-C₆alkylamino;-   R² is —OR¹¹, —SR¹¹, —NHR¹¹, or —N(R¹¹)₂;    -   where each R¹¹ independently is hydrogen, C₁-C₂₀ alkyl, C₂-C₂₀        alkenyl, C₂-C₂₀ alkynyl, or halo(C₁-C₂₀ alkyl);-   R³ is hydrogen, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, or    —OR⁷, each optionally substituted with one to four substituents    selected from halogen, —CN, —NO₂, C₁-C₆ alkyl, halo(C₁-C₆ alkyl),    —OR⁸, —NR⁸ ₂, —CO₂R⁸, —CONR⁸ ₂, C₃-C₈ cycloalkyl, C₃-C₈    cycloalkenyl, aryl, heteroaryl, and heterocycle, wherein each    cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle are    optionally substituted with R⁹;-   R⁴ and R⁵ are independently C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀    alkynyl, each optionally substituted with one to four substituents    selected from halogen, —CN, —NO₂, C₁-C₆ alkyl, halo(C₁-C₆ alkyl),    —OR⁸, —NR⁸ ₂, —CO₂R⁸, —CONR⁸ ₂, C₃-C₈ cycloalkyl, C₃-C₈    cycloalkenyl, aryl, heteroaryl, and heterocycle, wherein each    cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle are    optionally substituted with R⁹;-   each R⁶ is hydrogen, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl,    C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, aryl, heteroaryl, heterocycle,    or —OR¹⁰, wherein each alkyl, alkenyl, and alkynyl are optionally    substituted with one to four substituents selected from halogen,    —CN, —NO₂, C₁-C₆ alkyl, halo(C₁-C₆ alkyl), —OR⁸, —NR⁸ ₂, —CO₂R⁸,    —CONR⁸ ₂, C₃-C₈ cycloalkyl optionally substituted with R⁹, C₃-C₈    cycloalkenyl optionally substituted with R⁹, aryl optionally    substituted with R⁹, heteroaryl optionally substituted with R⁹, and    heterocycle optionally substituted with R⁹;    -   where R¹⁰ is C₁-C₆ alkyl, halo(C₁-C₆ alkyl), C₃-C₈ cycloalkyl,        aryl, heteroaryl, heterocycle, aryl(C₁-C₆ alkyl),        C₃-C₈cycloalkyl(C₁-C₆ alkyl), aryl(C₁-C₆ alkyl),        heteroaryl(C₁-C₆ alkyl), or heterocycle(C₁-C₆ alkyl), wherein        each cycloalkyl, aryl, heteroaryl, and heterocycle are        optionally substituted with R⁹.

In one embodiment, the disclosure provides compounds of formula (II),wherein X is Br or Cl.

In one embodiment, the disclosure provides compounds as described abovewith reference to formula (II), wherein R¹ is hydrogen, optionallysubstituted C₁-C₂₀ alkyl, optionally substituted C₂-C₂₀ alkenyl,optionally substituted C₂-C₂₀ alkynyl, or —OR⁷. In one embodiment, R¹ ishydrogen.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (II), wherein R¹ is optionally substitutedC₁-C₂₀ alkyl. In one embodiment, C₁-C₂₀ alkyl is optionally substitutedwith —OR⁸, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, or aryl, wherein eachcycloalkyl, cycloalkenyl, and aryl are optionally substituted with R⁹.In another embodiment, R¹ is C₁-C₂₀ alkyl optionally substituted withC₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, or aryl, wherein each cycloalkyl,cycloalkenyl, and aryl are optionally substituted with R⁹.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (II), wherein R¹ is —OR⁷ and R⁷ is C₁-C₆alkyl.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (II), wherein R² is —OR¹¹. In anotherembodiment, R² is —OR¹¹, and R¹¹ is hydrogen, C₁-C₆ alkyl, or halo(C₁-C₆alkyl).

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (II), wherein R² is —OH.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (II), wherein R² is —NHR¹¹, or —N(R¹¹)₂.In another embodiment, R² is —NHR¹¹ or —N(R¹¹)₂, and R¹¹ is hydrogen,C₄-C₂₀ alkyl, C₄-C₂₀ alkenyl, or C₄-C₂₀ alkynyl.

In another embodiment, the disclosure provides compounds as describedabove with any reference to formula (II), wherein R² is —N(R¹¹)₂. In yetanother embodiment, R² is —N(R¹¹)₂, and R¹¹ is hydrogen, C₄-C₂₀ alkyl,C₄-C₂₀ alkenyl, or C₄-C₂₀ alkynyl. In a further embodiment, R² is—N(R¹¹)₂, and R¹¹ is hydrogen or C₄-C₂₀ alkyl. Even further, R² is—N(C₄-C₂₀ alkyl)₂.

In another embodiment, the disclosure provides compounds as describedabove with any reference to formula (II), wherein R² is —N(decyl)₂.

In another embodiment, the disclosure provides compounds as describedabove with any reference to formula (II), wherein R² is —N(pentyl)₂.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (II), wherein R³ is hydrogen, optionallysubstituted C₁-C₂₀ alkyl, or —OR⁷. In one embodiment, R³ is hydrogen. Inanother embodiment R³ is optionally substituted C₁-C₂₀ alkyl. In yetanother embodiment, C₁-C₂₀ alkyl is optionally substituted with —OR⁸,C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, or aryl, wherein each cycloalkyl,cycloalkenyl, and aryl are optionally substituted with R⁹.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (II), wherein R³ is C₁-C₂₀ alkyloptionally substituted with C₃-C₅ cycloalkyl, C₃-C₈ cycloalkenyl, oraryl, wherein each cycloalkyl, cycloalkenyl, and aryl are optionallysubstituted with R⁹.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (II), wherein R³ is —OR⁷ and R⁷ is C₁-C₆alkyl.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (II), wherein R⁴ is optionally substitutedC₁-C₂₀ alkyl. In one embodiment, R⁴ is C₁-C₂₀ alkyl.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (II), wherein R⁵ is optionally substitutedC₁-C₂₀ alkyl. In one embodiment, R⁵ is C₁-C₂₀ alkyl.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (II), wherein R⁶ is hydrogen or optionallysubstituted C₁-C₂₀ alkyl. In one embodiment, R⁶ is hydrogen. In anotherembodiment, R⁶ is C₁-C₂₀ alkyl optionally substituted with —OR⁸, C₃-C₈cycloalkyl, C₃-C₈ cycloalkenyl, or aryl, wherein each cycloalkyl,cycloalkenyl, and aryl are optionally substituted with R⁹. In yetanother embodiment, C₁-C₂₀ alkyl is optionally substituted with C₃-C₈cycloalkyl, C₃-C₈ cycloalkenyl, or aryl, wherein each cycloalkyl,cycloalkenyl, and aryl are optionally substituted with R⁹.

In one embodiment, the disclosure provides compounds as described abovewith reference to formula (II), wherein R⁶ is —OR¹⁰, where R¹⁰ is C₁-C₆alkyl, C₃-C₈ cycloalkyl, aryl, heteroaryl, heterocycle, aryl(C₁-C₆alkyl), C₃-C₈ cycloalkyl(C₁-C₆ alkyl), aryl(C₁-C₆ alkyl),heteroaryl(C₁-C₆ alkyl), or heterocycle(C₁-C₆ alkyl), wherein eachcycloalkyl, aryl, heteroaryl, and heterocycle are optionally substitutedwith R⁹. In another embodiment, R⁶ is —OR¹⁰, where R¹⁰ is aryl oraryl(C₁-C₆ alkyl), where each aryl is optionally substituted with R⁹.

In one embodiment, the disclosure provides compounds as described abovewith reference to formula (II), wherein X is another anion other thanhalogen. Suitable anions include, but are not limited to, carbonate,hydrogen carbonate, hydroxide, nitrate, nitrite, cyanide, phosphate,sulfate, sulfite, acetate, formate, propionate, isopropionate, malonate,maleate, lacate, succiniate, tartrate, citrate and oxalate.

In one embodiment, the disclosure provides compounds of formula (II)wherein R² is —OH, and R³-R⁶ are as described above with any referenceto formula (II). In another embodiment, these compounds can tautomerizeas follows:

Therefore, another aspect of the disclosure provides compounds offormula (II-A)

wherein

-   R³ is hydrogen, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, or    —OR⁷, each optionally substituted with one to four substituents    selected from halogen, —CN, —NO₂, C₁-C₆ alkyl, halo(C₁-C₆ alkyl),    —OR⁸, —NR⁸ ₂, —CO₂R⁸, —CONR⁸ ₂, C₃-C₈ cycloalkyl, C₃-C₈    cycloalkenyl, aryl, heteroaryl, and heterocycle, wherein each    cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle are    optionally substituted with R⁹;    -   where each R⁷ independently is hydrogen, C₁-C₆ alkyl, or        halo(C₁-C₆ alkyl);    -   where each R⁸ independently is hydrogen, C₁-C₆ alkyl, halo(C₁-C₆        alkyl), C₃-C₈ cycloalkyl, aryl, heteroaryl, heterocycle,        aryl(C₁-C₆ alkyl), C₃-C₈ cycloalkyl(C₁-C₆ alkyl), aryl(C₁-C₆        alkyl), heteroaryl(C₁-C₆ alkyl), or heterocycle(C₁-C₆ alkyl),        wherein each cycloalkyl, aryl, heteroaryl, and heterocycle are        optionally substituted with R⁹;    -   where each R⁹ independently is halogen, —CN, —NO₂, —N₃, C₁-C₆        alkyl, halo(C₁-C₆ alkyl), C₁-C₆ alkoxy, amino, C₁-C₆alkylamino,        or diC₁-C₆alkylamino;-   R⁴ and R⁵ are independently C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀    alkynyl, each optionally substituted with one to four substituents    selected from halogen, —CN, —NO₂, C₁-C₆ alkyl, halo(C₁-C₆ alkyl),    —OR⁸, —NR⁸ ₂, —CO₂R⁸, —CONR⁸ ₂, C₃-C₈ cycloalkyl, C₃-C₈    cycloalkenyl, aryl, heteroaryl, and heterocycle, wherein each    cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle are    optionally substituted with R⁹;-   each R⁶ is hydrogen, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl,    C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, aryl, heteroaryl, or    heterocycle, wherein each alkyl, alkenyl, and alkynyl are optionally    substituted with one to four substituents selected from halogen,    —CN, —NO₂, C₁-C₆ alkyl, halo(C₁-C₆ alkyl), —OR⁸, —NR⁸ ₂, —CO₂R⁸,    —CONR⁸ ₂, C₃-C₈ cycloalkyl optionally substituted with R⁹, C₃-C₈    cycloalkenyl optionally substituted with R⁹, aryl optionally    substituted with R⁹, heteroaryl optionally substituted with R⁹, and    heterocycle optionally substituted with R⁹.

In one embodiment, the disclosure provides compounds of formula (II-A),wherein R³ is hydrogen, optionally substituted C₁-C₂₀ alkyl, or —OR⁷. Inone embodiment, R³ is hydrogen. In another embodiment R³ is optionallysubstituted C₁-C₂₀ alkyl. In yet another embodiment, C₁-C₂₀ alkyl isoptionally substituted with —OR⁸, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl,or aryl, wherein each cycloalkyl, cycloalkenyl, and aryl are optionallysubstituted with R⁹.

In one embodiment, the disclosure provides compounds of formula (II-A),wherein R³ is C₁-C₂₀ alkyl optionally substituted with C₃-C₈ cycloalkyl,C₃-C₈ cycloalkenyl, or aryl, wherein each cycloalkyl, cycloalkenyl, andaryl are optionally substituted with R⁹.

In one embodiment, the disclosure provides compounds of formula (II-A),wherein R³ is —OR⁷ and R⁷ is C₁-C₆ alkyl.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (II-A), wherein R⁴ is optionallysubstituted C₁-C₂₀ alkyl. In one embodiment, R⁴ is C₁-C₂₀ alkyl.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (II-A), wherein R⁵ is optionallysubstituted C₁-C₂₀ alkyl. In one embodiment, R⁵ is C₁-C₂₀ alkyl.

In one embodiment, the disclosure provides compounds as described abovewith any reference to formula (II-A), wherein R⁶ is hydrogen oroptionally substituted C₁-C₂₀ alkyl. In one embodiment, R⁶ is hydrogen.In another embodiment, R⁶ is C₁-C₂₀ alkyl optionally substituted with—OR⁸, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, or aryl, wherein eachcycloalkyl, cycloalkenyl, and aryl are optionally substituted with R⁹.In yet another embodiment, C₁-C₂₀ alkyl is optionally substituted withC₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, or aryl, wherein each cycloalkyl,cycloalkenyl, and aryl are optionally substituted with R⁹.

In one embodiment, the disclosure provides compounds as described abovewith reference to formula (I) or (II), which can exist as prodrugs. Forexample, —OH group in formula (I) and —R² in formula (II) can exist asprodrug. Suitable prodrugs include alkyl and aryl esters, acetates,carbamates, amino acid esters, such as glycine and alanine, and thelike.

In another embodiment, the disclosure provides pharmaceuticallyacceptable salts of compounds of the disclosure. Generally,pharmaceutically acceptable salts are those salts that retainsubstantially one or more of the desired pharmacological activities ofthe parent compound and which are suitable for administration to humans.Pharmaceutically acceptable salts include acid addition salts formedwith inorganic acids or organic acids. Inorganic acids suitable forforming pharmaceutically acceptable acid addition salts include, by wayof example and not limitation, hydrohalide acids (e.g., hydrochloricacid, hydrobromic acid, hydriodic, etc.), sulfuric acid, nitric acid,phosphoric acid, and the like. Organic acids suitable for formingpharmaceutically acceptable acid addition salts include, by way ofexample and not limitation, acetic acid, monofluoroacetic acid,difluoroacetic acid, trifluoroacetic acid, propionic acid, hexanoicacid, cyclopentanepropionic acid, glycolic acid, oxalic acid, pyruvicacid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,alkylsulfonic acids (e.g., methanesulfonic acid, ethanesulfonic acid,1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, etc.),arylsulfonic acids (e.g., benzenesulfonic acid, 4-chlorobenzenesulfonicacid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,camphorsulfonic acid, etc.),4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoicacid, salicylic acid, stearic acid, muconic acid, and the like. Forexample, in one embodiment, the salt is a trifluoroacetic acid salt,p-toluenesulfonic acid salt, or hydrochloride salt.

Pharmaceutically acceptable salts also include salts formed when anacidic proton present in the parent compound is either replaced by aninorganic ion (e.g., an alkali metal ion such as Na⁺, K⁺ or Li⁺, analkaline earth ion such as Ca²⁺ or Mg²⁺, an aluminum ion, or an ammoniumion) or coordinates with an organic base (e.g., ethanolamine,diethanolamine, triethanolamine, N-methylglucamine, morpholine,piperidine, dimethylamine, diethylamine, etc.).

The compounds described herein, as well as the salts thereof, may alsobe in the form of hydrates, solvates and N-oxides, as are well-known inthe art.

Therapeutic Applications

In one aspect, the disclosure provides a method for treating orprotecting mitochondria with respiratory chain lesions, comprisingadministering to a subject in need of such treatment an effective amountof one or more compounds of the invention.

Compounds of the disclosure are useful, for example, for treating orsuppressing diseases associated with decreased mitochondrial functionresulting in diminished ATP production and/or oxidative stress and/orlipid peroxidation in a subject in need of treatment. The presentdisclosure provides methods of treating conditions including but notlimited to Friedreich's ataxia, Leber's Hereditary Optic Neuropathy,Kearns-Sayre Syndrome, Mitochondrial Encephalomyopathy with LacticAcidosis and Stroke-Like Episodes, and Leigh syndrome in a subject byadministering an effective amount of a compound as described above withrespect to any of formulae (I), (II), or (II-A), including a salt orsolvate or stereoisomer thereof.

The disclosure also provides methods of treating conditions includingbut not limited to obesity, atherosclerosis, amyotrophic lateralsclerosis, Parkinson's Disease, cancer, heart failure, myocardialinfarction (MI), Alzheimer's Disease, Huntington's Disease,schizophrenia, bipolar disorder, fragile X syndrome, chronic fatiguesyndrome, and Leigh syndrome, in a subject by administering an effectiveamount of a compound as described above with respect to any of formulae(I), (II), or (II-A), including a salt or solvate or stereoisomerthereof.

In addition, the compounds of the invention can be used for prophylaxisof redox stress and enhancement of cellular function.

Friedreich's Ataxia

Friedreich's ataxia is a severe neurodegenerative and cardiodegenerativecondition. It is characterized by progressive ataxia of the limbs,muscle weakness, dysarthria, skeletal deformities and cardiomyopathy.While the biochemical basis of the disease is still under investigation,it is strongly associated with insufficient frataxin (Wilson et al.(1997) Nat. Genet. 16, 352-357; Wilson et al. (2003) J. Neurol. Sci.207, 103-105). In the majority of patients the insufficiency of frataxinis a consequence of an intronic GAA triplet repeat expansion in the genefor frataxin, which results in a significant decrease in its mRNAlevels, and ultimately in protein levels as well (Campuzano et al.(1996) Science 271, 1423-1427; Campuzano et al. (1997) Hum. Mol. Genet.6, 1771-1780). Frataxin acts as an iron chaperone during hemebiosynthesis (Bencze et al. (2007) J.C.S. Chem. Commun. 1798-1800) andhas been shown to be capable of stimulating the in vitro assembly ofheme and Fe—S clusters (Park et al. (2003) J. Biol. Chem. 278,31340-31351; Yoon et al. (2003) J. Am Chem. Soc. 125, 6078-6084; Yoon etal. (2004) J. Biol. Chem. 279, 25943-25946). Frataxin can interactphysically with mitochondrial electron transport chain proteins, as wellas with mitochondrial aconitase (which contains an Fe—S cluster)(Bulteau et al. (2004) Science 305, 242-245; Gonzalez-Cabo et al. (2005)Hum. Mol. Genet. 14, 2091-2098). Therefore, frataxin deficiency resultsin disruption of cellular iron homeostasis, with a progressive ironaccumulation in the mitochondrion, and a deficiency in heme and Fe—Sclusters.

It is believed that a deficiency in frataxin leads to compromisedmitochondrial respiratory chain function through a failure to assembleone or more Fe-utilizing proteins; one or more Fe—S clusters in themitochondrial respiratory complexes are likely to represent a criticallocus. In fact, diminished function of these complexes has been noted inFriedreich's ataxia patients (Bradley et al. (2000) Hum. Mol. Genet. 9,275-282). The loss of mitochondrial respiratory chain function can leadto diminished ATP production, while the accumulation of Fe in themitochondria makes the organelle highly susceptible to oxidative damageby reactive oxygen species, whose concentration increases concomitantwith the decrease in respiratory chain function. There is compellingevidence that while oxidative damage is not the primary lesion inFriedreich's ataxia, oxidative stress helps to drive diseaseprogression. Therefore, strategies to overcome oxidative stress shouldblunt disease progression and provide effective therapy.

Other Exemplary Mitochondrial Diseases

Leber hereditary optic neuropathy is associated with degeneration ofretinal ganglion cells and causes progressive loss of vision resultingin various degrees of blindness. Leber hereditary optic neuropathyprimarily affects men over the age of 20 and is maternally transmitteddue to mutations in the mitochondrial (not nuclear) genome.

Kearns-Sayre syndrome is a rare neuromuscular disorder typically withonset usually before the age of 20. It is characterized by progressiveexternal ophthalmoplegia (paralysis of the eye muscles) and mildskeletal muscle weakness, hearing loss, loss of coordination, heartproblems, and cognitive delays. There are many other names for theKearns-Sayre syndrome including: Chronic progressive externalophthalmoplegia CPEO with myopathy; CPEO with ragged-red fibers; KSS;Mitochondrial cytopathy, Kearns-Sayre type; Oculocraniosomatic syndrome;Ophthalmoplegia-plus syndrome; Ophthalmoplegia with myopathy; andOphthalmoplegia with ragged-red fibers.

Mitochondrial Encephalomyopathy with Lactic Acidosis and Stroke-LikeEpisodes is a progressive mitochondrial disease that involves multipleorgan systems including the central nervous system, cardiac muscle,skeletal muscle, and gastrointestinal system. Symptoms include muscleweakness, stroke-like events, eye muscle paralysis, and cognitiveimpairment. Leigh syndrome is a degenerative brain disorder is usuallydiagnosed at a young age (e.g. before age two). Deterioration is oftenrapid with symptoms such as seizures, dementia, feeding and speechdifficulties, respiratory dysfunction, heart problems, and muscleweakness. Prognosis is poor with death typically occurring within a fewyears of diagnosis.

Mitochondrial Energy Production

Energy released from the citric acid (Krebs) cycle in the mitochondrialmatrix enters the mitochondrial electron transport chain as NADH(complex I) and FADH₂ (complex II). These are the first two of fiveprotein complexes involved in ATP production, all of which are locatedin the inner mitochondrial membrane. Electrons derived from NADH (byoxidation with a NADH-specific dehydrogenase) and FADH₂ (by oxidationwith succinate dehydrogenase) travel down the respiratory chain,releasing their energy in discrete steps by driving the active transportof protons from the mitochondrial matrix to the intermembrane space(i.e., through the inner mitochondrial membrane). The electron carriersin the respiratory chain include flavins, protein-bound iron-sulfurcenters, quinones, cytochromes and copper. There are two molecules thattransfer electrons between complexes: coenzyme Q (complex I→III, andcomplex II→III) and cytochrome c (complex III→IV). The final electronacceptor in the respiratory chain is O₂, which is converted to H₂O incomplex IV.

In a functional mitochondrion, transport of two electrons throughcomplex I results in the transport of 4H⁺ into the intermembrane space.Two more H⁺ transfers to the intermembrane space result from electrontransport through complex III, and four more H⁺ transfers from electrontransport through complex IV. The 10 electrons transported to theintermembrane space create a proton electrochemical gradient; they canreturn to the mitochondrial matrix via complex V (ATP synthase), withthe concomitant conversion of ADP to ATP. It is interesting that no H⁺is transferred to the intermembrane space as a consequence of electrontransport through complex II. Therefore, 2e⁻ transfer from FADH₂(complex II→complex III→complex IV) results in the transport of only 6protons, compared with 10 protons resulting from 2e⁻ transfer from NADH(complex I→complex III→complex IV), with correspondingly less ATPproduced. Each glucose molecule metabolized by glycolysis produces 12electrons; these are converted to 5 NADH molecules and 1 FADH₂ via theKrebs cycle in the mitochondrial matrix. The 5 NADH molecules employedin mitochondrial electron transport produce about 25 ATPs, while thesingle FADH₂ affords only about 3 ATP molecules. (There are another 4molecules of ATP derived from glucose metabolism—2 during glycolysis and2 in the Krebs cycle). While this analysis underscores the importance ofcomplex I involvement in normal ATP production, it also tends to obscurecertain metabolic realities/uncertainties that may offer importantopportunities for therapeutic intervention. One metabolic reality isthat complex I, while important quantitatively for ATP production innormal mitochondria, is not essential for all mitochondrial ATPproduction. Electrons can enter the electron transport chain at thelevel of coenzyme Q (either from complex II or from fatty acidoxidation), producing about 60% as much ATP as would have resulted hadthey entered the electron transport chain at complex I). While the fluxof electrons that normally enter the individual mitochondrial complexes,ultimately passing through coenzyme Q, is probably dictated largely bythe availability of electrons derived from NADH, FADH₂ and fatty acidoxidation, the actual intrinsic capacity of the individual pathways doesnot appear to have been studied carefully.

In functional mitochondria, a few experimental parameters can bemeasured readily, reflecting mitochondrial respiration. These includeNADH and O₂ consumption, and ATP production. Less readily measured arethe electrons that flow through the electron transport chain, therebyconsuming oxygen, and producing H₂O and ATP. The electrons within themitochondria can really only be measured when they are associated withone of the mitochondrial electron carriers such as coenzyme Q. Inhumans, this mitochondrial coenzyme is present as coenzyme Q₁₀, whichhas a 50-carbon C-substituent that renders the molecule virtuallyinsoluble in water (calculated octanol-water partition coefficient>10²⁰) (James et al. (2005) J Biol. Chem. 280, 21295-21312).

In dysfunctional mitochondria, one can still carry out the same types ofmeasurements as noted above for functioning mitochondria. If the flow ofelectrons through complex I is interrupted, several measured parametersshould change. These include diminished consumption of NADH (measured asincreased lactate through pyruvate reduction) and diminished ATPproduction. Since electrons will not flow as efficiently from complex Ito coenzyme Q, the concentration of this reduced coenzyme will diminish.Interestingly, a new pathway for oxygen consumption is created. Whileoxygen is not converted as efficiently to water in complex IV (anoverall four electron reduction of each oxygen molecule), much of theflow of electrons into a defective complex I is redirected to oxygen,with the production of superoxide (a one electron reduction of eachoxygen). Thus, the stoichiometry of oxygen utilization is altered. Theproduction of superoxide by mitochondria actually occurs to some extenteven in normal mitochondria, but is a much more frequent event inmitochondria containing defects in the respiratory chain. Superoxide isone form of reactive oxygen species (ROS). Superoxide itself is notbelieved to react readily with biological molecules such lipidmembranes, proteins and DNA, and actually functions as a signalingmolecule for the regulation of certain cellular processes. Biologically,the main fate of superoxide (O^(−.) ₂) is a disproportionation reactionwith itself to produce peroxide (H₂O₂) and oxygen, i.e.2O^(−.) ₂+2H⁺→H₂O₂+O₂This reaction occurs spontaneously, and can also be catalyzed bysuperoxide dismutase. Superoxide can also be reduced to peroxide in amonovalent process. Like superoxide, hydrogen peroxide is also notintrinsically deleterious to cellular macromolecules, and is actuallyessential to the function of a number of enzymes. However, in thepresence of metal ions such as iron and copper, hydrogen peroxide isconverted to hydroxyl radical (HO.) and hydroxide ion (OH⁻) according tothe Fenton reaction, i.e.HOOH+Fe²⁺→Fe³⁺+HO.+OH⁻Hydroxyl radicals are very highly reactive, capable of reacting withvirtually any biological molecule, including DNA, proteins and lipids.Hydroxyl radicals can also diffuse through cells readily, and theirability to damage cells is limited only by the distance that they travelbefore they react. Hydroxyl radicals can also react with superoxide,producing singlet oxygen ((¹O₂)+OH⁻), another highly reactive form ofROS that damages cellular macromolecules and assemblies. Oneparticularly deleterious and well-studied reaction mediated by hydroxylradicals is the abstraction of hydrogen atoms (H.) from membrane lipids,forming a carbon-centered radical (R.). This radicalHO.+RH(lipid)→R.+H₂OR.+O₂→ROO.ROO.+RH→ROOH+R.can readily react with oxygen, forming a hydroperoxy radical (ROO.). Thehydroperoxy radical is also highly reactive, and can abstract anotherhydrogen atom from the membrane lipid, producing another carbon-centeredradical (which can undergo precisely the same chemistry), ultimatelyproducing a chain reaction affording many oxidative lesions in themembrane lipids from a single hydroxyl radical (lipid peroxidation). Itis for this reason that lipid peroxidation likely represents a majorprocess by which cellular and mitochondrial membranes are degraded incells containing (partially) dysfunctional mitochondria. The observedaccumulation of lipofuscin in Friedreich's ataxia patients is fullyconsistent with the thesis that lipid peroxidation is a central processthat drives disease progression (La Marche et al. (1980) Can. J.Neurosci. 7, 389-396; Yin, D. (1996) Free Rad. Biol. Med. 21, 871-888;Yamada et al. (2001) J. Lipid Res. 42, 1187-1196).

It may be noted that while all lesions in the mitochondrial electrontransport chain that affect mitochondrial dysfunction will result inelevated levels of superoxide, some types of lesions may be expected toproduce more functional damage. The latter would certainly includeFriedreich's ataxia, in which suboptimal levels of the protein frataxin(which is responsible for cellular iron homeostasis; Park et al. (2003)J. Biol. Chem. 278, 31340-31351; Yoon et al. (2003) J. Am. Chem. Soc.125, 6078-6084; Yoon et al. (2004) J. Biol. Chem. 279, 25943-25946;Bencze et al. (2007) J.C.S. Chem. Commun. 1798-1800) and assembly of FeSclusters in proteins results in an accumulation of Fe^(2+/3+) within themitochondria, and contributes instead to the Fenton chemistry notedabove. Likewise, disorders such as amyotrophic lateral sclerosis areassociated with a deficiency in the detoxifying enzyme superoxidedismutase, and will have greatly enhanced concentrations of the ROSdiscussed above.

One poorly studied parameter of mitochondrial electron transport iswhether the process is best characterized as involving one or twoelectron transfers. The is important because NADH is an obligatorytwo-electron donor, and coenzyme Q and cytochrome c participate intwo-electron redox cycles, as does FADH₂. Virtually all publicationsrepresent the processes in which these species participate as involvinga net two electron change. However, FADH₂ may (and generally does)transfer its reducing equivalents as single electrons. Further, the Qcycle in complex III clearly involves single-electron transfers. Reducedcytochrome c is known to transfer electrons one at a time to cytochromec oxidase, the enzyme responsible for the final step in respiration.Finally, the accumulation of electrons within dysfunctional mitochondria(producing reductive stress) is relieved substantially by (one-electron)reduction of oxygen to superoxide (vide supra). Thus, while the electrontransport chain has the capacity to transfer two electrons by virtue ofthe redox cycles of most of its participants, it is not clear that itnecessarily must do so to function.

Given that the reductive stress (build-up of electrons) encounteredinitially in mitochondrial dysfunction is a one electron process, as islipid peroxidation, carriers of single electrons could find utility indealing with reductive stress, e.g. molecules in which the one-electronreduced intermediate is stabilized by dipole interactions, substituenteffects, resonance effects or captodative effects. Molecules designed totraffic single electrons, and which can (i) accept electrons fromsuperoxide (ii) donate electrons to complex III and (iii) quenchcarbon-centered lipid radicals are especially useful. MultifunctionalRadical Quenchers (MRQs) of the invention can effectively protectmitochondria, cells and organisms from oxidative stress.

Pharmaceutical Compositions

In another aspect, the present disclosure provides compositionscomprising one or more of compounds as described above with respect toany of formulae (I), (II), or (II-A) and an appropriate carrier,excipient or diluent. The exact nature of the carrier, excipient ordiluent will depend upon the desired use for the composition, and mayrange from being suitable or acceptable for veterinary uses to beingsuitable or acceptable for human use. The composition may optionallyinclude one or more additional compounds.

When used to treat or prevent such diseases, the compounds describedherein may be administered singly, as mixtures of one or more compoundsor in mixture or combination with other agents useful for treating suchdiseases and/or the symptoms associated with such diseases. Thecompounds may also be administered in mixture or in combination withagents useful to treat other disorders or maladies, such as steroids,membrane stabilizers, 5LO inhibitors, leukotriene synthesis and receptorinhibitors, inhibitors of IgE isotype switching or IgE synthesis, IgGisotype switching or IgG synthesis, β-agonists, tryptase inhibitors,aspirin, COX inhibitors, methotrexate, anti-TNF drugs, retuxin, PD4inhibitors, p38 inhibitors, PDE4 inhibitors, and antihistamines, to namea few. The compounds may be administered in the form of compounds perse, or as pharmaceutical compositions comprising a compound.

Pharmaceutical compositions comprising the compound(s) may bemanufactured by means of conventional mixing, dissolving, granulating,dragee-making levigating, emulsifying, encapsulating, entrapping orlyophilization processes. The compositions may be formulated inconventional manner using one or more physiologically acceptablecarriers, diluents, excipients or auxiliaries which facilitateprocessing of the compounds into preparations which can be usedpharmaceutically.

The compounds may be formulated in the pharmaceutical composition perse, or in the form of a hydrate, solvate, N-oxide or pharmaceuticallyacceptable salt, as previously described. Typically, such salts are moresoluble in aqueous solutions than the corresponding free acids andbases, but salts having lower solubility than the corresponding freeacids and bases may also be formed.

Pharmaceutical compositions may take a form suitable for virtually anymode of administration, including, for example, topical, ocular, oral,buccal, systemic, nasal, injection, transdermal, rectal, vaginal, etc.,or a form suitable for administration by inhalation or insufflation.

For topical administration, the compound(s) may be formulated assolutions, gels, ointments, creams, suspensions, etc. as are well-knownin the art. Systemic formulations include those designed foradministration by injection, e.g., subcutaneous, intravenous,intramuscular, intrathecal or intraperitoneal injection, as well asthose designed for transdermal, transmucosal oral or pulmonaryadministration.

Useful injectable preparations include sterile suspensions, solutions oremulsions of the active compound(s) in aqueous or oily vehicles. Thecompositions may also contain formulating agents, such as suspending,stabilizing and/or dispersing agent. The formulations for injection maybe presented in unit dosage form, e.g., in ampules or in multidosecontainers, and may contain added preservatives. Alternatively, theinjectable formulation may be provided in powder form for reconstitutionwith a suitable vehicle, including but not limited to sterile pyrogenfree water, buffer, dextrose solution, etc., before use. To this end,the active compound(s) may be dried by any art-known technique, such aslyophilization, and reconstituted prior to use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants are knownin the art.

For oral administration, the pharmaceutical compositions may take theform of, for example, lozenges, tablets or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulfate). The tablets may be coated by methods well known in theart with, for example, sugars, films or enteric coatings.

Liquid preparations for oral administration may take the form of, forexample, elixirs, solutions, syrups or suspensions, or they may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol, Cremophore™ or fractionated vegetable oils); and preservatives(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). Thepreparations may also contain buffer salts, preservatives, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the compound, as is well known.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For rectal and vaginal routes of administration, the compound(s) may beformulated as solutions (for retention enemas) suppositories orointments containing conventional suppository bases such as cocoa butteror other glycerides.

For nasal administration or administration by inhalation orinsufflation, the compound(s) can be conveniently delivered in the formof an aerosol spray from pressurized packs or a nebulizer with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons, carbondioxide or other suitable gas. In the case of a pressurized aerosol, thedosage unit may be determined by providing a valve to deliver a meteredamount. Capsules and cartridges for use in an inhaler or insufflator(for example capsules and cartridges comprised of gelatin) may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

For ocular administration, the compound(s) may be formulated as asolution, emulsion, suspension, etc. suitable for administration to theeye. A variety of vehicles suitable for administering compounds to theeye are known in the art.

For prolonged delivery, the compound(s) can be formulated as a depotpreparation for administration by implantation or intramuscularinjection. The compound(s) may be formulated with suitable polymeric orhydrophobic materials (e.g., as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, e.g., as asparingly soluble salt. Alternatively, transdermal delivery systemsmanufactured as an adhesive disc or patch which slowly releases thecompound(s) for percutaneous absorption may be used. To this end,permeation enhancers may be used to facilitate transdermal penetrationof the compound(s).

Alternatively, other pharmaceutical delivery systems may be employed.Liposomes and emulsions are well-known examples of delivery vehiclesthat may be used to deliver compound(s). Certain organic solvents suchas dimethylsulfoxide (DMSO) may also be employed, although usually atthe cost of greater toxicity.

The pharmaceutical compositions may, if desired, be presented in a packor dispenser device which may contain one or more unit dosage formscontaining the compound(s). The pack may, for example, comprise metal orplastic foil, such as a blister pack. The pack or dispenser device maybe accompanied by instructions for administration.

The compound(s) described herein, or compositions thereof, willgenerally be used in an amount effective to achieve the intended result,for example in an amount effective to treat or prevent the particulardisease being treated. By therapeutic benefit is meant eradication oramelioration of the underlying disorder being treated and/or eradicationor amelioration of one or more of the symptoms associated with theunderlying disorder such that the patient reports an improvement infeeling or condition, notwithstanding that the patient may still beafflicted with the underlying disorder. Therapeutic benefit alsogenerally includes halting or slowing the progression of the disease,regardless of whether improvement is realized.

The amount of compound(s) administered will depend upon a variety offactors, including, for example, the particular indication beingtreated, the mode of administration, whether the desired benefit isprophylactic or therapeutic, the severity of the indication beingtreated and the age and weight of the patient, the bioavailability ofthe particular compound(s) the conversation rate and efficiency intoactive drug compound under the selected route of administration, etc.

Determination of an effective dosage of compound(s) for a particular useand mode of administration is well within the capabilities of thoseskilled in the art. Effective dosages may be estimated initially from invitro activity and metabolism assays. For example, an initial dosage ofcompound for use in animals may be formulated to achieve a circulatingblood or serum concentration of the metabolite active compound that isat or above an IC₅₀ of the particular compound as measured in as invitro assay. Calculating dosages to achieve such circulating blood orserum concentrations taking into account the bioavailability of theparticular compound via the desired route of administration is wellwithin the capabilities of skilled artisans. Initial dosages of compoundcan also be estimated from in vivo data, such as animal models. Animalmodels useful for testing the efficacy of the active metabolites totreat or prevent the various diseases described above are well-known inthe art Animal models suitable for testing the bioavailability and/ormetabolism of compounds into active metabolites are also well-known.Ordinarily skilled artisans can routinely adapt such information todetermine dosages of particular compounds suitable for humanadministration.

Dosage amounts will typically be in the range of from about 0.0001mg/kg/day, 0.001 mg/kg/day or 0.01 mg/kg/day to about 100 mg/kg/day, butmay be higher or lower, depending upon, among other factors, theactivity of the active metabolite compound, the bioavailability of thecompound, its metabolism kinetics and other pharmacokinetic properties,the mode of administration and various other factors, discussed above.Dosage amount and interval may be adjusted individually to provideplasma levels of the compound(s) and/or active metabolite compound(s)which are sufficient to maintain therapeutic or prophylactic effect. Forexample, the compounds may be administered once per week, several timesper week (e.g., every other day), once per day or multiple times perday, depending upon, among other things, the mode of administration, thespecific indication being treated and the judgment of the prescribingphysician. In cases of local administration or selective uptake, such aslocal topical administration, the effective local concentration ofcompound(s) and/or active metabolite compound(s) may not be related toplasma concentration. Skilled artisans will be able to optimizeeffective local dosages without undue experimentation.

DEFINITIONS

The following terms and expressions used herein have the indicatedmeanings.

Terms used herein may be preceded and/or followed by a single dash, “-”,or a double dash, “=”, to indicate the bond order of the bond betweenthe named substituent and its parent moiety; a single dash indicates asingle bond and a double dash indicates a double bond. In the absence ofa single or double dash it is understood that a single bond is formedbetween the substituent and its parent moiety; further, substituents areintended to be read “left to right” unless a dash indicates otherwise.For example, C₁-C₆alkoxycarbonyloxy and —OC(O)C₁-C₆alkyl indicate thesame functionality; similarly arylalkyl and -alkylaryl indicate the samefunctionality.

The term “alkenyl” as used herein, means a straight or branched chainhydrocarbon containing from 2 to 10 carbons, unless otherwise specified,and containing at least one carbon-carbon double bond. Representativeexamples of alkenyl include, but are not limited to, ethenyl,2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl,2-heptenyl, 2-methyl-1-heptenyl, 3-decenyl, and3,7-dimethylocta-2,6-dienyl.

The term “alkoxy” as used herein, means an alkyl group, as definedherein, appended to the parent molecular moiety through an oxygen atom.Representative examples of alkoxy include, but are not limited to,methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, andhexyloxy.

The term “alkyl” as used herein, means a straight or branched chainhydrocarbon containing from 1 to 10 carbon atoms unless otherwisespecified. Representative examples of alkyl include, but are not limitedto, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, andn-decyl. When an “alkyl” group is a linking group between two othermoieties, then it may also be a straight or branched chain; examplesinclude, but are not limited to —CH₂—, —CH₂CH₂—, —CH₂CH₂CHC(CH₃)—,—CH₂CH(CH₂CH₃)CH₂—.

The term “alkynyl” as used herein, means a straight or branched chainhydrocarbon group containing from 2 to 10 carbon atoms and containing atleast one carbon-carbon triple bond. Representative examples of alkynylinclude, but are not limited, to acetylenyl, 1-propynyl, 2-propynyl,3-butynyl, 2-pentynyl, and 1-butynyl.

The term “aryl,” as used herein, means a phenyl (i.e., monocyclic aryl),or a bicyclic ring system containing at least one phenyl ring or anaromatic bicyclic ring containing only carbon atoms in the aromaticbicyclic ring system. The bicyclic aryl can be azulenyl, naphthyl, or aphenyl fused to a monocyclic cycloalkyl, a monocyclic cycloalkenyl, or amonocyclic heterocyclyl. The bicyclic aryl is attached to the parentmolecular moiety through any carbon atom contained within the phenylportion of the bicyclic system, or any carbon atom with the napthyl orazulenyl ring. The fused monocyclic cycloalkyl or monocyclicheterocyclyl portions of the bicyclic aryl are optionally substitutedwith one or two oxo and/or thia groups. Representative examples of thebicyclic aryls include, but are not limited to, azulenyl, naphthyl,dihydroinden-1-yl, dihydroinden-2-yl, dihydroinden-3-yl,dihydroinden-4-yl, 2,3-dihydroindol-4-yl, 2,3-dihydroindol-5-yl,2,3-dihydroindol-6-yl, 2,3-dihydroindol-7-yl, inden-1-yl, inden-2-yl,inden-3-yl, inden-4-yl, dihydronaphthalen-2-yl, dihydronaphthalen-3-yl,dihydronaphthalen-4-yl, dihydronaphthalen-1-yl,5,6,7,8-tetrahydronaphthalen-1-yl, 5,6,7,8-tetrahydronaphthalen-2-yl,2,3-dihydrobenzofuran-4-yl, 2,3-dihydrobenzofuran-5-yl,2,3-dihydrobenzofuran-6-yl, 2,3-dihydrobenzofuran-7-yl,benzo[d][1,3]dioxol-4-yl, benzo[d][1,3]dioxol-5-yl,2H-chromen-2-on-5-yl, 2H-chromen-2-on-6-yl, 2H-chromen-2-on-7-yl,2H-chromen-2-on-8-yl, isoindoline-1,3-dion-4-yl,isoindoline-1,3-dion-5-yl, inden-1-on-4-yl, inden-1-on-5-yl,inden-1-on-6-yl, inden-1-on-7-yl, 2,3-dihydrobenzo[b][1,4]dioxan-5-yl,2,3-dihydrobenzo[b][1,4]dioxan-6-yl, 2H-benzo[b][1,4]oxazin3(4H)-on-5-yl, 2H-benzo[b][1,4]oxazin3 (4H)-on-6-yl,2H-benzo[b][1,4]oxazin3 (4H)-on-7-yl, 2H-benzo[b][1,4]oxazin3(4H)-on-8-yl, benzo[d]oxazin-2(3H)-on-5-yl,benzo[d]oxazin-2(3H)-on-6-yl, benzo[d]oxazin-2(3H)-on-7-yl,benzo[d]oxazin-2(3H)-on-8-yl, quinazolin-4(3H)-on-5-yl,quinazolin-4(3H)-on-6-yl, quinazolin-4(3H)-on-7-yl,quinazolin-4(3H)-on-8-yl, quinoxalin-2(1H)-on-5-yl,quinoxalin-2(1H)-on-6-yl, quinoxalin-2(1H)-on-7-yl,quinoxalin-2(1H)-on-8-yl, benzo[d]thiazol-2(3H)-on-4-yl,benzo[d]thiazol-2(3H)-on-5-yl, benzo[d]thiazol-2(3H)-on-6-yl, and,benzo[d]thiazol-2(3H)-on-7-yl. In certain embodiments, the bicyclic arylis (i) naphthyl or (ii) a phenyl ring fused to either a 5 or 6 memberedmonocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, or a 5or 6 membered monocyclic heterocyclyl, wherein the fused cycloalkyl,cycloalkenyl, and heterocyclyl groups are optionally substituted withone or two groups which are independently oxo or thia.

The terms “cyano” and “nitrile” as used herein, mean a —CN group.

The term “cycloalkyl” as used herein, means a monocyclic or a bicycliccycloalkyl ring system. Monocyclic ring systems are cyclic hydrocarbongroups containing from 3 to 8 carbon atoms, where such groups can besaturated or unsaturated, but not aromatic. In certain embodiments,cycloalkyl groups are fully saturated. Examples of monocycliccycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. Bicycliccycloalkyl ring systems are bridged monocyclic rings or fused bicyclicrings. Bridged monocyclic rings contain a monocyclic cycloalkyl ringwhere two non-adjacent carbon atoms of the monocyclic ring are linked byan alkylene bridge of between one and three additional carbon atoms(i.e., a bridging group of the form —(CH₂)_(w)—, where w is 1, 2, or 3).Representative examples of bicyclic ring systems include, but are notlimited to, bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane,bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane, andbicyclo[4.2.1]nonane. Fused bicyclic cycloalkyl ring systems contain amonocyclic cycloalkyl ring fused to either a phenyl, a monocycliccycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or amonocyclic heteroaryl. The bridged or fused bicyclic cycloalkyl isattached to the parent molecular moiety through any carbon atomcontained within the monocyclic cycloalkyl ring. Cycloalkyl groups areoptionally substituted with one or two groups which are independentlyoxo or thia. In certain embodiments, the fused bicyclic cycloalkyl is a5 or 6 membered monocyclic cycloalkyl ring fused to either a phenylring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 memberedmonocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a5 or 6 membered monocyclic heteroaryl, wherein the fused bicycliccycloalkyl is optionally substituted by one or two groups which areindependently oxo or thia.

The term “halo” or “halogen” as used herein, means —Cl, —Br, —I or —F.

The term “haloalkyl” as used herein, means at least one halogen, asdefined herein, appended to the parent molecular moiety through an alkylgroup, as defined herein. Representative examples of haloalkyl include,but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl,pentafluoroethyl, and 2-chloro-3-fluoropentyl.

The term “heteroaryl,” as used herein, means a monocyclic heteroaryl ora bicyclic ring system containing at least one heteroaromatic ring. Themonocyclic heteroaryl can be a 5 or 6 membered ring. The 5 membered ringconsists of two double bonds and one, two, three or four nitrogen atomsand optionally one oxygen or sulfur atom. The 6 membered ring consistsof three double bonds and one, two, three or four nitrogen atoms. The 5or 6 membered heteroaryl is connected to the parent molecular moietythrough any carbon atom or any nitrogen atom contained within theheteroaryl. Representative examples of monocyclic heteroaryl include,but are not limited to, furyl, imidazolyl, isoxazolyl, isothiazolyl,oxadiazolyl, oxazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl,pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl,triazolyl, and triazinyl. The bicyclic heteroaryl consists of amonocyclic heteroaryl fused to a phenyl, a monocyclic cycloalkyl, amonocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclicheteroaryl. The fused cycloalkyl or heterocyclyl portion of the bicyclicheteroaryl group is optionally substituted with one or two groups whichare independently oxo or thia. When the bicyclic heteroaryl contains afused cycloalkyl, cycloalkenyl, or heterocyclyl ring, then the bicyclicheteroaryl group is connected to the parent molecular moiety through anycarbon or nitrogen atom contained within the monocyclic heteroarylportion of the bicyclic ring system. When the bicyclic heteroaryl is amonocyclic heteroaryl fused to a phenyl ring, then the bicyclicheteroaryl group is connected to the parent molecular moiety through anycarbon atom or nitrogen atom within the bicyclic ring system.Representative examples of bicyclic heteroaryl include, but are notlimited to, benzimidazolyl, benzofuranyl, benzothienyl, benzoxadiazolyl,benzoxathiadiazolyl, benzothiazolyl, cinnolinyl,5,6-dihydroquinolin-2-yl, 5,6-dihydroisoquinolin-1-yl, furopyridinyl,indazolyl, indolyl, isoquinolinyl, naphthyridinyl, quinolinyl, purinyl,5,6,7,8-tetrahydroquinolin-2-yl, 5,6,7,8-tetrahydroquinolin-3-yl,5,6,7,8-tetrahydroquinolin-4-yl, 5,6,7,8-tetrahydroisoquinolin-1-yl,thienopyridinyl, 4,5,6,7-tetrahydrobenzo[c][1,2,5]oxadiazolyl, and6,7-dihydrobenzo[c][1,2,5]oxadiazol-4(5H)-onyl. In certain embodiments,the fused bicyclic heteroaryl is a 5 or 6 membered monocyclic heteroarylring fused to either a phenyl ring, a 5 or 6 membered monocycliccycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 memberedmonocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl,wherein the fused cycloalkyl, cycloalkenyl, and heterocyclyl groups areoptionally substituted with one or two groups which are independentlyoxo or thia.

The term “heterocyclyl” as used herein, means a monocyclic heterocycleor a bicyclic heterocycle. The monocyclic heterocycle is a 3, 4, 5, 6 or7 membered ring containing at least one heteroatom independentlyselected from the group consisting of O, N, and S where the ring issaturated or unsaturated, but not aromatic. The 3 or 4 membered ringcontains 1 heteroatom selected from the group consisting of O, N and S.The 5 membered ring can contain zero or one double bond and one, two orthree heteroatoms selected from the group consisting of O, N and S. The6 or 7 membered ring contains zero, one or two double bonds and one, twoor three heteroatoms selected from the group consisting of O, N and S.The monocyclic heterocycle is connected to the parent molecular moietythrough any carbon atom or any nitrogen atom contained within themonocyclic heterocycle. Representative examples of monocyclicheterocycle include, but are not limited to, azetidinyl, azepanyl,aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl,1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl,isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl,oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl,piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl,pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl,thiadiazolidinyl, thiazolinyl, thiazolidinyl,thiomorpholinyl,1,1-dioxidothiomorpholinyl (thiomorpholine sulfone),thiopyranyl, and trithianyl. The bicyclic heterocycle is a monocyclicheterocycle fused to either a phenyl, a monocyclic cycloalkyl, amonocyclic cycloalkenyl, a monocyclic heterocycle, or a monocyclicheteroaryl. The bicyclic heterocycle is connected to the parentmolecular moiety through any carbon atom or any nitrogen atom containedwithin the monocyclic heterocycle portion of the bicyclic ring system.Representative examples of bicyclic heterocyclyls include, but are notlimited to, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzofuran-3-yl,indolin-1-yl, indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2-yl,decahydroquinolinyl, decahydroisoquinolinyl, octahydro-1H-indolyl, andoctahydrobenzofuranyl. Heterocyclyl groups are optionally substitutedwith one or two groups which are independently oxo or thia. In certainembodiments, the bicyclic heterocyclyl is a 5 or 6 membered monocyclicheterocyclyl ring fused to phenyl ring, a 5 or 6 membered monocycliccycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 memberedmonocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl,wherein the bicyclic heterocyclyl is optionally substituted by one ortwo groups which are independently oxo or thia.

The term “nitro” as used herein, means a —NO₂ group.

The term “oxo” as used herein means a ═O group.

The term “saturated” as used herein means the referenced chemicalstructure does not contain any multiple carbon-carbon bonds. Forexample, a saturated cycloalkyl group as defined herein includescyclohexyl, cyclopropyl, and the like.

The term “thia” as used herein means a ═S group.

The term “unsaturated” as used herein means the referenced chemicalstructure contains at least one multiple carbon-carbon bond, but is notaromatic. For example, a unsaturated cycloalkyl group as defined hereinincludes cyclohexenyl, cyclopentenyl, cyclohexadienyl, and the like.

“Pharmaceutically acceptable” refers to those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problems or complications commensurate with a reasonablebenefit/risk ratio or which have otherwise been approved by the UnitedStates Food and Drug Administration as being acceptable for use inhumans or domestic animals.

“Pharmaceutically acceptable salt” refers to both acid and base additionsalts.

“Prodrug” refers to compounds that are transformed (typically rapidly)in vivo to yield the parent compound of the above formulae, for example,by hydrolysis in blood. Common examples include, but are not limited to,ester and amide forms of a compound having an active form bearing acarboxylic acid moiety. Examples of pharmaceutically acceptable estersof the compounds of this invention include, but are not limited to,alkyl esters (for example with between about one and about six carbons)wherein the alkyl group is a straight or branched chain. Acceptableesters also include cycloalkyl esters and arylalkyl esters such as, butnot limited to benzyl. Examples of pharmaceutically acceptable amides ofthe compounds of this invention include, but are not limited to, primaryamides, and secondary and tertiary alkyl amides (for example withbetween about one and about six carbons). Amides and esters of thecompounds of the present invention may be prepared according toconventional methods. A thorough discussion of prodrugs is provided inT. Higuchi and V. Stella, “Pro-drugs as Novel Delivery Systems,” Vol 14of the A.C.S. Symposium Series, and in Bioreversible Carriers in DrugDesign, ed. Edward B. Roche, American Pharmaceutical Association andPergamon Press, 1987, both of which are incorporated herein by referencefor all purposes.

“Therapeutically effective amount” refers to that amount of a compoundwhich, when administered to a subject, is sufficient to effect treatmentfor a disease or disorder described herein. The amount of a compoundwhich constitutes a “therapeutically effective amount” will varydepending on the compound, the disorder and its severity, and the age ofthe subject to be treated, but can be determined routinely by one ofordinary skill in the art.

“Modulating” or “modulate” refers to the treating, prevention,suppression, enhancement or induction of a function, condition ordisorder. For example, it is believed that the compounds of the presentinvention can modulate atherosclerosis by stimulating the removal ofcholesterol from atherosclerotic lesions in a human.

“Treating” or “treatment” as used herein covers the treatment of adisease or disorder described herein, in a subject, preferably a human,and includes:

i. inhibiting a disease or disorder, i.e., arresting its development;

ii. relieving a disease or disorder, i.e., causing regression of thedisorder;

iii. slowing progression of the disorder; and/or

iv. inhibiting, relieving, or slowing progression of one or moresymptoms of the disease or disorder

“Subject” refers to a warm blooded animal such as a mammal, preferably ahuman, or a human child, which is afflicted with, or has the potentialto be afflicted with one or more diseases and disorders describedherein.

“EC₅₀” refers to a dosage, concentration or amount of a particular testcompound that elicits a dose-dependent response at 50% of maximalexpression of a particular response that is induced, provoked orpotentiated by the particular test compound.

“IC₅₀” refers to an amount, concentration or dosage of a particular testcompound that achieves a 50% inhibition of a maximal response in anassay that measures such response.

“Respiratory chain lesions” in mitochondria or “Mitochondria withrespiratory chain lesions” refers to mitochondria in which thestructures of the five complexes responsible for ATP production byoxidative phosphorylation are altered structurally, typically in a waythat leads to diminished function.

Methods of Synthesis

Many general references providing commonly known chemical syntheticschemes and conditions useful for synthesizing the disclosed compoundsare available (see, e.g., Smith and March, March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001; or Vogel, A Textbook of Practical OrganicChemistry, Including Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978).

Compounds as described herein can be purified by any of the means knownin the art, including chromatographic means, such as HPLC, preparativethin layer chromatography, flash column chromatography and ion exchangechromatography. Any suitable stationary phase can be used, includingnormal and reversed phases as well as ionic resins. Most typically thedisclosed compounds are purified via silica gel and/or aluminachromatography. See, e.g., Introduction to Modern Liquid Chromatography,2nd Edition, ed. L. R. Snyder and J. J. Kirkland, John Wiley and Sons,1979; and Thin Layer Chromatography, ed E. Stahl, Springer-Verlag, NewYork, 1969.

During any of the processes for preparation of the subject compounds, itmay be necessary and/or desirable to protect sensitive or reactivegroups on any of the molecules concerned. This may be achieved by meansof conventional protecting groups as described in standard works, suchas J. F. W. McOmie, “Protective Groups in Organic Chemistry”, PlenumPress, London and New York 1973, in T. W. Greene and P. G. M. Wuts,“Protective Groups in Organic Synthesis”, Third edition, Wiley, New York1999, in “The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer),Academic Press, London and New York 1981, in “Methoden der organischenChemie”, Houben-Weyl, 4.sup.th edition, Vol. 15/1, Georg Thieme Verlag,Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, “Aminosauren, Peptide,Proteine”, Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982,and/or in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharide andDerivate”, Georg Thieme Verlag, Stuttgart 1974. The protecting groupsmay be removed at a convenient subsequent stage using methods known fromthe art.

Representative synthetic procedures for the preparation of compounds ofthe invention are outlined below.

EXAMPLES

The compounds and methods of the disclosure are illustrated further bythe following examples, which are provided for illustrative purposes andare not intended to be construed as limiting the disclosure in scope orspirit to the specific compounds and methods described in them.

Example 1 Preparation of3-heptyl-1-hydroxy-2,6-dimethyl-5-vinylpyridin-4(1H)-one

3-heptyl-1-hydroxy-2,6-dimethyl-5-vinylpyridin-4(1H)-one (CPD-1)

To a mixture of 49 g of polyphosphoric acid and 30 mL of acetic acid wasadded 0.6 mL (0.5 g, 2.9 mmol) of 2-decanone. The resulting solution washeated at 130° C. with vigorous stirring for 8 h. The resulting darkblack solution was cooled to 0° C. and 100 mL of water was added. Themixture was extracted with portions of ethyl ether. The combined organicphase was washed with 10% aqueous KOH, dried over anhydrous MgSO₄ andconcentrated under diminished pressure to afford a crude residue. Theresidue was purified by chromatography on a silica gel column. Elutionwith hexane/ethyl acetate gave 3-heptyl-2,6-dimethyl-4H-pyran-4-one (1)as a light yellow solid: yield 555 mg (54%); silica gel TLC R_(f) 0.45(1:1 hexane/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 0.45-0.50 (m, 3H),0.88-1.04 (m, 10H), 1.82 (s, 3H), 1.89 (s, 3H), 1.98 (t, 2H, J=7.6 Hz),and 5.62 (s, 1H); ^(13C) NMR (CDCl₃) δ 13.3, 16.6, 18.9, 21.9, 23.6,27.8, 28.5, 28.9, 31.1, 112.1, 124.0, 160.6, 163.5, and 178.4.

Compound 1 (0.01 g, 30.6 mmol) was heated at 100° C. with aqueousammonia in a high pressure tube for 18 h. After the mixture was cooledto room temperature, the crude was concentrated under diminishedpressure. The residue was purified by chromatography on a silica gelcolumn. Elution with 5:1 ethyl acetate/methanol gave3-heptyl-2,6-dimethylpyridin-4(1H)-one (3) as a brown solid: yield 0.083g (81%); silica gel TLC R_(f) 0.25 (3:1 ethyl acetate/methanol); ¹H NMR(CD₃OD) δ 0.68-0.71 (m, 3H), 1.09-1.28 (m, 10H), 2.20 (s, 3H), 2.27 (s,3H), 2.34-2.36 (m, 2H) and 6.29 (s, 1H).

To a solution of 3 (100 mg, 0.45 mmol) in acetonitrile (2.5 mL) wasadded ceric ammonium nitrate (25 mg, 0.045 mmol) followed by iodine (126mg, 0.495 mmol). The reaction mixture was stirred at 70° C. undernitrogen for 7 h. After completion of the reaction, the mixture wascooled to room temperature and treated with an ice-cold aqueous solutionof sodium thiosulfate with stirring. The mixture was extracted withportions of ethyl ether. The combined organic phase was dried overanhydrous MgSO₄ and concentrated under diminished pressure to afford acrude residue. The residue was purified by chromatography on a silicagel column. Elution with hexane/ethyl acetate gave3-heptyl-5-iodo-2,6-dimethylpyridin-4(1H)-one (5) as a light yellowsolid: yield 119 mg (74%); silica gel TLC R_(f) 0.45 (1:1 hexane/ethylacetate); ¹H NMR (400 MHz, CDCl₃) δ 0.88-0.91 (m, 3H), 1.29-1.35 (m,10H), 2.33 (s, 3H) and 2.54-2.56 (m, 5H).

A mixture of t-BuOK (102 mg, 0.912 mmol) and 5 (0.06 g, 0.73 mmol) inanhydrous THF was stirred for 1 hr at room temperature Boc₂O (175 mg,0.80 mmol) was added and the mixture was heated at 60° C. for 1 h. Afterthe mixture was cooled to room temperature, the crude was concentratedunder diminished pressure to afford a yellow oil. The residue waspurified by chromatography on a silica gel column. Elution with 1:1hexane/ethyl acetate gave 3-iodo-5-heptyl-2,6-dimethylpyridin-4-yltert-butyl carbonate (7) as a light yellow oil: yield 61 mg (76%);silica gel TLC R_(f) 0.7 (1:1 hexane/ethyl acetate); ¹H NMR (CDCl₃) δ0.83-0.86 (m, 3H), 1.22-1.32 (m, 10H), 1.52 (s, 9H), 2.35 (s, 3H) and2.51-2.54 (m, 5H).

To a solution of 7 (60 mg, 0.864 mmol) in 1,4-dioxane (4 ml) were addedlithium chloride (73.2 mg, 1.73 mmol),tetrakis(triphenylphosphine)palladium (99.8 mg, 10 mol %) andtributyl(vinyl)tin (0.327 ml, 1.12 mmol). The mixture was heated atreflux for 3 h then cooled and diluted with CHCl₃. The mixture waswashed with brine, dried over Na₂SO₄, filtered, and concentrated inunder diminished pressure. The residue was purified by columnchromatography on silica gel [AcOEt/MeOH (20:1)] to afford tert-butyl3-heptyl-2,6-dimethyl-5-vinylpyridin-4-yl carbonate (9) (30 mg, 64%). ¹HNMR (CDCl₃) δ 0.85-0.88 (m, 3H), 1.27-1.32 (m, 10H), 1.47 (s, 9H), 2.40(s, 3H), 2.43 (s, 3H), 2.47-2.50 (m, 2H), 5.50 (dd, 2H, J=18 Hz, 11.2Hz) and 6.45 (dd, 1H, J=11.6 Hz, 11.6 Hz).

A solution of 9 (30 mg, 0.66 mmol) and mCPBA (125 mg, 0.72 mmol) inanhydrous CH₂Cl₂ (2 mL) was stirred at 0° C. for 1 hr under argonatmosphere. The crude was concentrated under diminished pressure. Theresidue was purified by chromatography on a silica gel column. Elutionwith 1:1 hexane/ethyl acetate gavetert-butyl-3-heptyl-2,6-dimethyl-5-vinylpyridin-4-yl-carbonate-N-oxide(11) as a light yellow oil: yield 24 mg (77%); silica gel TLC R_(f) 0.25(ethyl acetate); ¹H NMR (CDCl₃) δ 0.85-0.88 (m, 3H), 1.26-1.32 (m, 10H),1.50 (s, 9H), 2.50 (s, 3H), 2.52 (s, 3H), 2.55-2.57 (m, 2H), 5.56 (dd,2H, J=18 Hz, 11.2 Hz) and 6.54 (dd, 1H, J=11.6 Hz, 11.6 Hz).

Aqueous 10 M KOH was added to a solution of 11 (138 mg, 0.54 mmol) inEtOH (1.5 mL). After 12 hr at room temperature water (3 mL) was addedand pH was brought to 1-2 using concentrated HCl, The mixture wasextracted with portions of ethyl ether. The combined organic phase wasdried over anhydrous MgSO₄ and concentrated under diminished pressure toafford a crude residue. Elution with 5:1 chloroform/methanol gave3-heptyl-1-hydroxy-2,6-dimethyl-5-vinylpyridin-4(1H)-one (CPD-1) as alight yellow oil: yield 12 mg (71%); silica gel TLC R_(f) 0.35 (1:1ethyl acetate/methanol); ¹H NMR (CDCl₃) δ 0.87 (m, 3H), 1.26-1.39 (m,10H), 2.39 (s, 3H), 2.47 (s, 3H), 2.60 (m, 2H), 5.57 (dd, 2H, J=18 Hz,11.6 Hz) and 6.54 (dd, 1H, J=11.6 Hz, 11.2 Hz).

Example 2 Preparation of3-hexadecyl-1-hydroxy-2,6-dimethyl-5-vinylpyridin-4(1H)-one

3-hexadecyl-1-hydroxy-2,6-dimethyl-5-vinylpyridin-4(1H)-one (CPD-2)

To a mixture of 49 g of polyphosphoric acid and 30 mL of acetic acid wasadded 0.6 mL (0.5 g, 1.77 mmol) of 2-nonadecanone. The resultingsolution was heated at 130° C. with vigorous stirring for 8 h. Theresulting dark black solution was cooled to 0° C. and 100 mL of waterwas added. The mixture was extracted with portions of ethyl ether. Thecombined organic phase was washed with 10% aqueous KOH, dried overanhydrous MgSO₄ and concentrated under diminished pressure to afford acrude residue. The residue was purified by chromatography on a silicagel column. Elution with chloroform gave3-hexadecyl-2,6-dimethyl-4H-pyran-4-one (2) as a light yellow solid:yield 331 mg (50%); silica gel TLC R_(f) 0.6 (1:1 hexane/ethyl acetate);¹H NMR (CDCl₃, 400 MHz) δ 0.78-0.82 (m, 3H), 1.18-1.24 (m, 28H), 2.14(s, 3H), 2.21 (s, 3H), 2.30 (t, 2H, J=8 Hz) and 6.06 (s, 1H).

Compound 2 (0.02 g, 30.6 mmol) was heated at 100° C. with aqueousammonia in a high pressure tube for 18 h. After the mixture was cooledto room temperature, the crude was concentrated under diminishedpressure. The residue was purified by chromatography on a silica gelcolumn. Elution with 5:1 ethyl acetate/methanol gave3-hexadecyl-2,6-dimethylpyridin-4(1H)-one (4) as a brown solid; yield0.01 g (51%); silica gel TLC R_(f) 0.25 (3:1 ethyl acetate/methanol); ¹HNMR (CDCl₃, 400 MHz) δ 0.88-0.89 (m, 3H), 1.28-1.32 (m, 28H), 2.28 (s,3H), 2.34 (s, 3H), 2.44-2.47 (m, 2H) and 6.18 (s, 1H).

To a solution of 4 (100 mg, 4.52 mmol) in acetonitrile (25 mL) was addedceric ammonium nitrate (0.248 g, 0.45 mmol) followed by iodine (0.631 g,4.97 mmol). The mixture was stirred at 70° C. under nitrogen for 7 h.After completion of the reaction, the mixture was cooled to roomtemperature and treated with an ice-cold aqueous solution of sodiumthiosulfate with stirring. The mixture was extracted with portions ofethyl ether. The combined organic phase was dried over anhydrous MgSO₄and concentrated under diminished pressure to afford a crude residue.The residue was purified by chromatography on a silica gel column.Elution with hexane/ethyl acetate gave3-hexadecyl-5-iodo-2,6-dimethylpyridin-4(1H)-one (6) as a light yellowsolid: yield 93 mg (68%); silica gel TLC R_(f) 0.60 (1:1 hexane/ethylacetate); ¹H NMR (400 MHz, CDCl₃) δ 0.86-0.89 (m, 3H), 1.24-1.28 (m,28H), 2.20 (s, 3H), 2.26 (s, 3H) and 2.32-2.39 (m, 2H).

A mixture of t-BuOK (102 mg, 0.912 mmol) and2,3,6-trimethylpyridin-4(1H)-one(1) (0.1 g, 0.73 mmol) in anhydrous THFwas stirred for 1 hr at room temperature Boc₂O (175 mg, 0.80 mmol) wasadded and the mixture was heated at 60° C. for 1 h. After the mixturewas cooled to room temperature, the crude was concentrated underdiminished pressure to a yellow oil. The residue was purified bychromatography on a silica gel column. Elution with 1:1 hexane/ethylacetate gave tert-butyl 3-hexadecyl-5-iodo-2,6-dimethylpyridin-4-ylcarbonate (8) as a light yellow oil: yield 85 mg (75%); silica gel TLCR_(f) 0.85 (1:1 hexane/ethyl acetate); ¹H NMR (CDCl₃) δ 0.85-0.89 (m,3H), 1.25-1.31 (m, 28H), 1.57 (s, 9H), 2.50 (s, 3H), 2.52-2.56 (m, 2H)and 2.72 (s, 3H)

To a solution of 8 (85 mg, 0.864 mmol) in 1,4-dioxane (4 ml) were addedlithium chloride (73.2 mg, 1.73 mmol),tetrakis(triphenylphosphine)palladium (99.8 mg, 10 mol %) andtributyl(vinyl)tin (0.327 ml, 1.12 mmol). The mixture was heated atreflux for 3 h then cooled and diluted with CHCl₃. The mixture waswashed with brine, dried over Na₂SO₄, filtered, and concentrated inunder diminished pressure. The residue was purified by columnchromatography on silica gel [AcOEt/MeOH (20:1)] to afford tert-butyl3-hexadecyl-2,6-dimethyl-5-vinylpyridin-4-yl carbonate (10) (19 mg,27%). ¹H NMR (CDCl₃) δ 0.02-0.03 (m, 3H), 1.20-1.27 (m, 28H), 1.46 (s,9H), 2.45 (s, 3H), 2.48 (s, 3H), 2.54-2.56 (m, 2H), 5.55 (dd, 2H, J=17.6Hz, 11.6 Hz) and 6.50 (dd, 1H, J=11.2 Hz, 11.2 Hz).

A solution of 10 (30 mg, 0.66 mmol) and mCPBA (125 mg, 0.72 mmol) inanhydrous CH₂Cl₂ (2 mL) was stirred at 0° C. for 1 h under argonatmosphere. The crude product was concentrated under diminishedpressure. The residue was purified by chromatography on a silica gelcolumn. Elution with 1:1 hexane/ethyl acetate gavetert-butyl-3-hexadecyl-2,6-dimethyl-5-vinylpyridin-4-yl-carbonate-N-oxide(12) as a light yellow oil: yield 23 mg (76%); silica gel TLC R_(f) 0.45(ethyl acetate); ¹H NMR (CDCl₃) δ 0.015-0.03 (m, 3H), 1.17-1.20 (m,28H), 1.48 (s, 9H), 2.55 (s, 3H), 2.58 (s, 3H), 2.60-2.62 (m, 2H), 5.62(dd, 2H, J=17.6 Hz, 11.6 Hz) and 6.568 (dd, 1H, J=11.2 Hz, 11.2 Hz).

Aqueous 10 M KOH was added to a solution of 12 (23 mg, 0.54 mmol) inEtOH (1.5 mL). After 12 hr at room temperature, water (3 mL) was addedand pH was brought to 1-2 using concentrated HCl, The mixture wasextracted with portions of ethyl ether. The combined organic phase wasdried over anhydrous MgSO₄ and concentrated under diminished pressure toafford a crude residue. Elution with 5:1 chloroform/methanol gave3-hexadecyl-1-hydroxy-2,6-dimethyl-5-vinylpyridin-4(1H)-one (CPD-2) as alight yellow oil: yield 13 mg (76%); silica gel TLC R_(f) 0.25 (3:1ethyl acetate/methanol); ¹H NMR (CDCl₃) δ 0.02-0.04 (m, 3H), 1.17-1.24(m, 28H), 2.45 (s, 3H), 2.52 (s, 3H), 2.61-2.65 (m, 2H), 5.64 (dd, 2H,J=17.6 Hz, 11.2 Hz) and 6.57 (dd, 1H, J=11.6 Hz, 11.2 Hz).

Example 3 Preparation of3-amino-5-heptyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one

3-amino-5-heptyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one (CPD-3)

Premixed nitric acid (d 1.5, 0.15 g) and sulfuric acid (0.05 g) wasadded dropwise at 5-10° C. to 3 (94 mg) in sulfuric acid (d 1.64, 0.5g). The mixture was heated at 80° C. for 1 hr then cooled and added toice. Saturated aqueous sodium carbonate was added until effervescenceceased. The mixture was extracted with portions of ethyl ether. Thecombined organic phase was dried over anhydrous MgSO₄ and concentratedunder diminished pressure to afford a crude residue. Elution withhexane/ethyl acetate gave 3-heptyl-2,6-dimethyl-5-nitropyridin-4(1H)-one(13) as a yellow oil: yield 76 mg (67%); silica gel TLC R_(f) 0.45 (1:1hexane/ethyl acetate); ¹H NMR (400 MHz, CDCl₃) δ 0.77-0.81 (m, 3H),1.19-1.27 (m, 10H), 2.29 (s, 3H), 2.35 (s, 3H) and 2.44-2.47 (m, 2H).

A mixture of t-BuOK (40 mg, 0.36 mmol, 1.25) and 13 (76 mg, 0.29 mmol)in anhydrous THF was stirred for 1 hr at room temperature Boc₂O (63 mg,0.32 mmol) was added and the mixture was heated at 60° C. for 5 h. Afterthe mixture was cooled to room temperature, the crude product wasconcentrated under diminished pressure to afford a yellow oil. Theresidue was purified by chromatography on a silica gel column. Elutionwith 1:1 hexane/ethyl acetate gave tert-butyl3-heptyl-2,6-dimethyl-5-nitropyridin-4-yl carbonate (15) as a lightyellow oil; yield 81 mg (79%); silica gel TLC R_(f) 0.7 (1:1hexane/ethyl acetate); ¹H NMR (400 MHz, CDCl3) δ 0.79-0.83 (m, 3H),1.18-1.28 (m, 10H), 1.48 (s, 9H) and 2.48-2.51 (m, 8H).

A solution of 15 (28 mg, 0.077 mmol) and mCPBA (15 mg, 0.084 mmol) inanhydrous CH₂Cl₂ (2 mL) was stirred at 0° C. for 1 hr under argonatmosphere. The crude product was concentrated under diminishedpressure. The residue was purified by chromatography on a silica gelcolumn. Elution with 1:1 hexane/ethyl acetate gave tert-butyl3-heptyl-2,6-dimethyl-5-nitropyridin-4-yl carbonate-N-oxide (17) as alight brown solid; yield 18 mg (62%); silica gel TLC R_(f) 0.40 (ethylacetate); ¹H NMR (CDCl₃) δ 1.12 (s, 9H), 1.74 (s, 3H), 2.06 (s, 3H),2.10 (s, 3H) and 6.63 (s, 1H).

To a mixture of 15 mg of solid NaBH₄ (11 mg, 0.29 mmol) and 45 mg ofsulfur (32 mg, 1.012 mmol) was added 3 mL of anhydrous THF dropwise.After the mixture was stirred for 5 min, 18 mg of compound 17 (0.047mmol) was added in 1 mL of THF. The reaction mixture was heated atreflux for 1 h, the reaction mixture was cooled to room temperature, thecrude product was concentrated under diminished pressure to a affordyellow oil. The residue was purified by chromatography on a silica gelcolumn. Elution with 10:1 ethyl acetate/methanol gave3-amino-5-heptyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one (CPD-3) as alight yellow oil: yield 8 mg (74%); silica gel TLC R_(f) 0.25 (1:1 ethylacetate/methanol); ¹H NMR (CDCl₃) δ 0.86-0.90 (m, 3H), 1.25-1.32 (m,10H) and 2.35-2.45 (m, 8H).

Example 4 Preparation of3-amino-5-hexadecyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one

3-amino-5-hexadecyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one (CPD-4)

Premixed nitric acid (d 1.5, 0.15 g) and sulfuric acid (0.05 g) wasadded dropwise at 5-10° C. to 4 (58 mg) in sulphuric acid (d 1.64, 0.5g). The reaction mixture was heated at 80° C. for 1 hr then cooled andadded to ice. Saturated aqueous sodium carbonate was added untileffervescence ceased. The reaction mixture was extracted with portionsof ethyl ether. The combined organic phase was dried over anhydrousMgSO₄ and concentrated under diminished pressure to afford a cruderesidue Elution with hexane/ethyl acetate gave3-hexadecyl-2,6-dimethyl-5-nitropyridin-4(1H)-one (14) as a yellow oil:yield 41 mg (62%); silica gel TLC R_(f) 0.60 (1:1 hexane/ethyl acetate);¹H NMR (400 MHz, CDCl3) δ 0.82-0.85 (m, 3H), 1.21-1.29 (m, 28H), 2.33(s, 3H), 2.39 (s, 3H) and 2.46-2.52 (m, 2H).

A mixture of t-BuOK (13 mg, 0.113 mmol) and 14 (41 g, 0.08 mmol) inanhydrous THF was stirred for 1 hr at room temperature Boc₂O (20 mg,0.09 mmol) was added and the reaction mixture was heated at 60° C. for 5hr. After the mixture was cooled to room temperature, the crude productwas concentrated under diminished pressure to afford a yellow oil. Theresidue was purified by chromatography on a silica gel column. Elutionwith 1:1 hexane/ethyl acetate gave tert-butyl3-hexadecyl-2,6-dimethyl-5-nitropyridin-4-yl carbonate (16) as a lightyellow oil: yield 27 mg (53%); silica gel TLC R_(f) 0.85 (1:1hexane/ethyl acetate); ¹H NMR (CDCl₃) δ 0.84-0.87 (m, 3H), 1.23-1.34 (m,28H), 1.52 (s, 9H), 2.44-2.50 (m, 2H) and 2.53-2.56 (s, 6H).

A solution of 16 (27 mg, 0.05 mmol) and mCPBA (10 mg, 0.06 mmol) inanhydrous CH₂Cl₂ (2 mL) was stirred at 0° C. for 1 hr under argonatmosphere. The crude product was concentrated under diminishedpressure. The residue was purified by chromatography on a silica gelcolumn. Elution with 1:1 hexane/ethyl acetate gave tert-butyl3-hexadecyl-2,6-dimethyl-5-nitropyridin-4-yl carbonate-N-oxide (18) as alight brown solid; yield 20 mg (72%); silica gel TLC R_(f) 0.50 (ethylacetate); ¹H NMR (CDCl₃) δ 0.84-0.88 (m, 3H), 1.22-1.30 (m, 28H), 1.54(s, 9H), 2.56-2.60 (m, 5H) and 2.62 (s, 3H).

To a mixture of 9.0 mg of solid NaBH₄ (0.24 mmol) and 21 mg of sulfur(0.646 mmol) was added 3 mL of anhydrous THF dropwise. After thereaction mixture was stirred for 5 min, 20 mg of 18 (0.03 mmol) wasadded in 1 mL of THF. The reaction mixture was heated at reflux for 1 h,the reaction mixture was cooled to room temperature, the crude productwas concentrated under diminished pressure to afford a yellow oil. Theresidue was purified by chromatography on a silica gel column. Elutionwith 10:1 ethyl acetate/methanol gave3-amino-5-hexadecyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one (CPD-4) as alight yellow oil: yield 11 mg (76%); silica gel TLC R_(f) 0.35 (1:1ethyl acetate/methanol); ¹H NMR (CDCl₃) δ 0.85-0.90 (m, 3H), 1.20-1.32(m, 28H) and 2.22-2.52 (m, 8H)

Example 5 Preparation of3-heptyl-1-hydroxy-5-methoxy-2,6-dimethylpyridin-4(1H)-one

3-heptyl-1-hydroxy-5-methoxy-2,6-dimethylpyridin-4(1H)-one (CPD-5)

Compound 5 (38 mg, 0.11 mmol) was added to a stirred suspension ofoil-free NaH (5 mg, 0.11 mmol) in DMF (5 mL). When evolution of hydrogenceased, benzyl chloride (14 mg, 0.11 mmol) was added and the mixture washeated at 50° C. for 4 h. The reaction mixture was added to water (5mL), extracted with portions of ethyl ether. The combined organic phasewas dried over anhydrous MgSO₄ and concentrated under diminishedpressure to afford a crude residue. Elution with 1:1 hexane/ethylacetate gave 4-(benzyloxy)-3-heptyl-5-iodo-2,6-dimethylpyridine (19) asa light yellow oil: yield 38 mg (80%); silica gel TLC R_(f) 0.7 (1:1hexane/ethyl acetate); ¹H NMR (CDCl₃) δ 0.61-0.89 (m, 3H), 1.26-1.31 (m,10H), 2.504 (s, 3H), 2.62-2.65 (m, 2H) 2.75 (s, 3H), 4.95 (s, 2H),7.38-7.43 (m, 4H) and 7.55-7.57 (m, 1H).

To a solution of sodium methoxide prepared from sodium (5.0 mg, 0.23mmol) and MeOH (1 mL) were added the iodo derivative 19 (27 mg, 0.06mmol) and CuI (1.66 mg, 0.009 mmol). The reaction mixture was heated at110° C. for 18 h and cooled, and 1 M aqueous NH4Cl was added. Themixture was extracted with portions of ethyl ether. The combined organicphase was dried over anhydrous MgSO₄ and concentrated under diminishedpressure to afford a crude residue. Elution with ethyl acetate gave4-(benzyloxy)-3-heptyl-5-methoxy-2,6-dimethylpyridine (20) as a lightyellow oil: yield 19 mg (71%); silica gel TLC R_(f) 0.3 (1:1hexane/ethyl acetate); ¹H NMR (CDCl₃) δ 0.60-0.89 (m, 3H), 1.26-1.30 (m,10H), 2.48 (s, 3H), 2.60-2.62 (m, 2H) 2.74 (s, 3H), 3.70 (s, 3H). 4.95(s, 2H), 7.38-7.42 (m, 4H) and 7.53-7.56 (m, 1H).

A solution of 20 (19 mg, 0.66 mmol) and mCPBA (125 mg, 0.72 mmol) inanhydrous CH₂Cl₂ (2 mL) was stirred at 0° C. for 1 hr under argonatmosphere, the solution was washed with aqueous NaHCO₃ dried overanhydrous MgSO₄ and concentrated under diminished pressure to afford acrude residue. The residue was dissolved in EtOH, Pd/C was added andafter applying an H₂ atmosphere (1 bar), the solution was stirred atroom temp for 30 min, After filtration through celite, which wasthoroughly washed with EtOH, the filtrate was concentrated underdiminished pressure and purified by chromatography on a silica gelcolumn. Elution with 20:1 ethyl acetate/methanol gave3-heptyl-1-hydroxy-5-methoxy-2,6-dimethylpyridin-4(1H)-one (CPD-5) as alight yellow oil; yield 9 mg (61%); silica gel TLC R_(f) 0.25 (3:1 ethylacetate/methanol); ¹H NMR (CDCl₃) δ 0.84-0.89 (m, 3H), 1.24-1.35 (m,10H), 2.45 (s, 3H), 2.50 (s, 3H) 2.63-2.67 (m, 2H) and 3.74 (s, 3H).

Example 6 Preparation of3-heptyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one

3-heptyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one (CPD-6)

A mixture of trimethyl pyrone 1 (113 mg, 0.51 mmol), hydroxylaminehydrochloride (707 mg, 10.2 mmol), sodium acetate (835 mg, 10.2 mmol),water (1 mL), and ethanol (2 mL) was heated to reflux for 8 hr. Afterthe reaction mixture was cooled to room temperature, filtered, and thefiltrate was concentrated under diminished pressure. The residue waspurified by chromatography on a silica gel column. Elution with 5:1ethyl acetate/methanol gave3-heptyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one (CPD-6): yield 63 mg(51%); silica gel TLC R_(f) 0.35 (3:1 ethyl acetate/methanol); ¹H NMR(400 MHz, CDCl3) δ 0.84-0.88 (m, 3H), 1.24-1.45 (m, 10H), 2.36 (s, 3H),2.47 (s, 3H), 2.60 (t, 2H, J=7.6 Hz) and 6.73 (s, 1H).

Example 7 Preparation of 3-decyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one

3-decyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one (CPD-7)

To a mixture of 49 g of polyphosphoric acid and 30 mL of acetic acid wasadded 0.6 mL (0.5 g, 2.53 mmol) of 2-tridecanone. The resulting solutionwas heated at 130° C. with vigorous stirring for 8 h. The resulting darkblack solution was cooled to 0° C. and 100 mL of water was added. Themixture was extracted with portions of ethyl ether. The combined organicphase was washed with 10% aqueous KOH, dried over anhydrous MgSO₄ andconcentrated under diminished pressure to afford a crude residue. Theresidue was purified by chromatography on a silica gel column. Elutionwith chloroform gave 3-decyl-2,6-dimethyl-4H-pyran-4-one (21) as a lightyellow solid: yield 331 mg (50%); silica gel TLC R_(f) 0.52 (1:1hexane/ethyl acetate); ¹H NMR (CDCl₃, 400 MHz) δ 0.52-0.55 (m, 3H),0.92-0.96 (m, 10H), 1.87 (s, 3H), 1.94 (s, 3H), 2.03 (t, 2H, J=7.2 Hz)and 5.73 (s, 1H); ^(13C) NMR (CDCl₃) δ 13.7, 16.9, 19.2, 22.3, 23.9,28.2, 28.9, 29.1, 29.2, 29.3, 31.6, 112.3, 124.3, 161.2, 164.1, and178.9.

A mixture of 21 (200 mg, 0.75 mmol), hydroxylamine hydrochloride (1.05g, 15.2 mmol), sodium acetate (1.24 g, 15.2 mmol), water (1 mL), andethanol (2 mL) was heated at reflux for 3 days. After the reactionmixture was cooled to room temperature, the crude was concentrated underdiminished pressure to afford a yellow oil. The residue was purified bychromatography on a silica gel column. Elution with 5:1chloroform/methanol gave 3-decyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one(CPD-7) as a light yellow oil: yield 0.08 g (40%); silica gel TLC R_(f)0.4 (3:1 ethyl acetate/methanol); ¹H NMR (CDCl₃, 400 MHz) δ 0.84-0.88(m, 3H), 1.24-1.44 (m, 16H), 2.27 (s, 3H), 2.48 (s, 3H), 2.57-2.61 (m,2H) and 6.65 (s, 1H).

Example 8 Preparation of3-hexadecyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one

3-hexadecyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one (CPD-8)

A mixture of 2 (200 mg, 0.57 mmol), hydroxylamine hydrochloride (399 mg,5.7 mmol), sodium acetate (471 mg, 5.7 mmol), water (1 mL), and ethanol(2 mL) was heated at reflux for 3 days. After the reaction mixture wascooled to room temperature, the crude product was concentrated underdiminished pressure to a yellow oil. The residue was purified bychromatography on a silica gel column. Elution with 5:1chloroform/methanol gave3-hexadecyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one (CPD-8) as a whitesolid: yield 0.088 g (42%); silica gel TLC R_(f) 0.5 (3:1 ethylacetate/methanol); ¹H NMR (CDCl₃, 400 MHz) δ 0.83-0.85 (m, 3H),1.22-1.27 (m, 28H), 2.32 (s, 3H), 2.46 (s, 3H), 2.55 (t, 2H, J=8 Hz) and6.7 (s, 1H).

Example 9 Preparation of3-bromo-5-heptyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one

3-bromo-5-heptyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one (CPD-9)

To a stirred solution of CPD-6 (63 mg, 0.266 mmol) in acetic acid (2 M)was added dropwise a solution of bromine (0.04 mL, 0.266 mmol) in aceticacid (1 mL) at 10-15° C. After 1.5 h a few drops of a saturated aqueoussodium sulfite solution was added to discharge excess bromine. Thereaction mixture was diluted with water and extracted with portions ofethyl ether. The combined organic phase was dried over anhydrous MgSO₄and concentrated under diminished pressure to afford a crude residue.The residue was purified by chromatography on a silica gel column toafford compound 3-bromo-5-heptyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one(CPD-9): yield 57 mg (69%); silica gel TLC R_(f) 0.68 (5:1 ethylacetate/methanol); ¹H NMR (CDCl₃) δ 0.82-0.86 (m, 3H), 1.23-1.36 (m,10H), 2.42 (s, 3H), 2.51-2.54 (m, 5H).

Example 10 Preparation of3-bromo-5-decyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one

3-bromo-5-decyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one (CPD-10)

To a stirred solution of CPD-7 (40 mg, 0.143 mmol) in acetic acid (2 M)was added dropwise a solution of bromine (0.02 mL, 0.143 mmol) in aceticacid (1 mL) at 10-15° C. After 1.5 h a few drops of a saturated aqueoussodium sulfite solution was added to discharge excess bromine. Thereaction mixture was diluted with water and extracted with portions ofethyl ether. The combined organic phase was dried over anhydrous MgSO₄and concentrated under diminished pressure to afford a crude residue.The residue was purified by chromatography on a silica gel column toafford compound 3-bromo-5-decyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one(CPD-10): yield 37 mg (73%); silica gel TLC R_(f) 0.75 (5:1 ethylacetate/methanol); ¹H NMR (CDCl₃, 400 MHz) δ 0.84-0.88 (m, 3H),1.24-1.44 (m, 16H), 2.36 (s, 3H) and 2.51-2.54 (m, 5H).

Example 11 Preparation of3-bromo-5-hexadecyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one

3-bromo-5-hexadecyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one (CPD-11)

To a stirred solution of CPD-8 (44 mg, 0.12 mmol) in acetic acid (2 M)was added dropwise a solution of bromine (0.006 mL, 0.12 mmol) in aceticacid (1 mL) at 10-15° C. After 1.5 h a few drops of a saturated aqueoussodium sulfite solution was added to discharge excess bromine. Thereaction mixture was diluted with water and extracted with portions ofethyl ether. The combined organic phase was dried over anhydrous MgSO₄and concentrated under diminished pressure to afford a crude residue.The residue was purified by chromatography on a silica gel column toafford compound3-bromo-5-hexadecyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one (CPD-11):yield 37 mg (69%); silica gel TLC R_(f) 0.80 (5:1 ethylacetate/methanol); ¹H NMR (CDCl₃, 400 MHz) δ 0.81-0.84 (m, 3H),1.21-1.29 (m, 28H), 2.37 (s, 3H) and 2.47-2.50 (m, 5H).

Example 12 Preparation of3-chloro-5-heptyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one

3-chloro-5-heptyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one (CPD-12)

To a stirred solution of CPD-6 (11 mg, 0.046 mmol) in acetic acid (10mL) was added N-chlorosuccinimide (6.7 g, 0.05 mmol). The mixture washeated at 100° C. for 8 hr and cooled to room temperature. The reactionmixture was diluted with water and extracted with portions of ethylether. The combined organic phase was dried over anhydrous MgSO₄ andconcentrated under diminished pressure to afford a crude residue. Theresidue was purified by chromatography on a silica gel column to affordcompound 3-chloro-5-heptyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one(CPD-12): yield 7 mg (54%); silica gel TLC R_(f) 0.66 (5:1 ethylacetate/methanol); ¹H NMR (400 MHz, CDCl₃) δ 0.77-0.81 (m, 3H),1.18-1.26 (m, 10H), 2.28 (s, 3H), 2.35 (s, 3H) and 2.44-2.47 (m, 2H).

Example 13 Preparation of3-chloro-5-decyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one

3-chloro-5-decyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one (CPD-13)

To a stirred solution of CPD-7 (40 mg, 0.143 mmol) in acetic acid (10mL) was added N-chlorosuccinimide (19 mg, 0.143 mmol). The reactionmixture was heated at 100° C. for 8 hr and cooled to room temperature.The reaction mixture was diluted with water and extracted with portionsof ethyl ether. The combined organic phase was dried over anhydrousMgSO₄ and concentrated under diminished pressure to afford a cruderesidue. The residue was purified by chromatography on a silica gelcolumn to afford compound3-chloro-5-decyl-1-hydroxy-2,6-dimethylpyridin-4(1H)-one (CPD-13): yield30 mg (67%); silica gel TLC R_(f) 0.7 (7:1 ethyl acetate/methanol); ¹HNMR (400 MHz, CDCl₃) δ 0.73-0.78 (m, 3H), 1.15-1.20 (m, 16H), 2.25 (s,3H), 2.30 (s, 3H) and 2.37-2.40 (m, 2H).

Example 14 Preparation of 3,7-bis(didecylamino)phenothiazin-5-iumbromide

3,7-bis(didecylamino)phenothiazin-5-ium bromide (CPD-14)

To a solution of 1.0 g (5.03 mmol) of phenothiazine in 60 mL of glacialacetic acid was added a solution 16.08 g (100.6 mmol) of bromine in 40mL of glacial acetic acid with vigorous stirring. After stirring forthree minutes, 400 mL of water was added to the reaction mixture. Theresulting red precipitate was filtered, washed with diethyl ether anddried under vacuum to give 3,7-dibromophenothiazin-5-ium bromide (22):yield 2.19 g (100%); mp 90° C. (dec.).

To a solution of 1.7 g (5.72 mmol) of didecylamine in 20 mL of CHCl₃ wasadded 0.63 g (1.43 mmol) of 22. The reaction mixture was stirred at roomtemperature for 2 h under argon and concentrated under diminishedpressure to give 3,7-bis(didecylamino)phenothiazin-5-ium bromide(CPD-14): mass spectrum (MALDI-TOF), m/z 788.69 (M⁺).

Example 15 Preparation of 3,7-bis(dipentylamino)phenothiazin-5-iumbromide

3,7-bis(dipentylamino)phenothiazin-5-ium bromide (CPD-15)

To a solution of 3.2 g (20.1 mmol) of dipentylamine in 60 mL of CHCl₃was added 4.37 g (10.03 mmol) of 22. The reaction mixture was stirred atr.t for 2 h under argon and concentrated under reduced pressure to give3,7-bis(dipentylamino)phenothiazin-5-ium bromide (CPD-15): mass spectrum(MALDI-TOF), m/z 509.38 (M+H)⁺.

Example 16 Preparation of 7-(didecylamino)-3H-phenothiazine-3-one

7-(didecylamino)-3H-phenothiazine-3-one (CPD-16)

To a solution of 0.13 g of CPD-14 in 5 mL of water and 5 mL of THF wasadded 0.034 g (0.2 mmol) of AgNO₃ dissolved in excess NH₄OH. Thereaction mixture was heated to reflux for an hour, filtered andextracted with two 10-mL portions of EtOAc. The violet organic layer waswashed with brine, dried over anhydrous Na₂SO₄, concentrated underdiminished pressure. The dark solid was purified on a silica gel column(30×1.25 cm). Elution with 1:1 hexanes/EtOAc gave CPD-16 as a darksolid: yield 2 mg (2.5%); silica gel TLC R_(f) 0.5 (1:1 hexanes/EtOAc);mass spectrum (MALDI-TOF), m/z 509.35 (M+H)⁺.

Example 17 Preparation of 7-(dipentylamino)-3H-phenothiazin-3-one

7-(dipentylamino)-3H-phenothiazin-3-one (CPD-17)

To a solution of 5.1 g of CPD-15 in 100 mL of water was added 1.9 g(11.2 mmol) of AgNO₃ dissolved in excess NH₄OH. The reaction mixture wasrefluxed for an hour, filtered and extracted with five 20-mL portions ofEtOAc. The violet organic layer was washed with brine, dried overanhydrous Na₂SO₄, concentrated under diminished pressure. The dark solidwas purified on a silica gel column (30×1.25 cm). Elution with EtOAcgave CPD-17 as a dark solid: yield 40 mg (<1%); silica gel TLC R_(f)0.57 (EtOAc); mass spectrum (MALDI-TOF), m/z 369.55 (M+H)⁺.

Example 18 Preparation of 7-(dimethylamino)-3H-phenothiazin-3-one

7-(dimethylamino)-3H-phenothiazin-3-one (CPD-18)

To a solution of 0.5 g (1.56 mmol) of Methylene blue in 50 mL of waterwas added 0.53 g (3.12 mmol) of AgNO₃ dissolved in excess NH₄OH. Thereaction mixture was heated to reflux for an hour, filtered andextracted with EtOAc (20×5 mL). The organic layer was washed with brine,dried over anhydrous Na₂SO₄, concentrated under diminished pressure. Thedark solid was purified on a silica gel column. Elution with 9:1EtOAc/MeOH gave CPD-18 as a dark solid: yield-25 mg (6%); silica gel TLCR_(f) 0.27 (9:1 EtOAc/MeOH); ¹H NMR (CDCl₃) δ 3.16 (s, 1H), 6.60 (d, 1H,J 2.8 Hz), 6.69 (d, 1H, J 2.4 Hz), 6.83 (d, 1H, J 2 Hz), 6.86 (d, 1H, J2.8 Hz), 7.67 (d, 1H, J 10 Hz), 7.71 (d, 1H, J 9.2 Hz); mass spectrum(MALDI-TOF), m/z 257.07 (M+H)⁺.

Example 19 Compounds of formula (I)

The following compounds may be prepared essentially according to theprocedures set forth above, with modifications where necessary of thestarting materials to provide the desired product:

Example 20 Compounds of Formula (II)

wherein:

n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14; and

Example 21 Cell Culture

Leukemic CEM cells (ATCC®, catalogue number CRL-2264) were cultured inRPMI (GIBCO, Grand island, NY, USA) with 10% FBS (Fisher Scientific, TX,USA), 2 mM glutamine (HyClone, South Logan, Utah, USA) and 1%penicillin-streptomycin mix antibiotics (Cellgro, Manassas, Va., USA)supplements. Cells were maintained in the log phase at a concentrationof between 1×10⁵ and 1×10⁶ cells/mL.

Friedreich's Ataxia lymphocytes and control cells (Coriell, cataloguenumber GM158150, and GM158151, respectively) were cultured in RPMI(GIBCO, Grand island, NY, USA) with 15% FBS (Fisher Scientific, TX,USA), 2 mM glutamine (HyClone, South Logan, Utah, USA) and 1%penicillin-streptomycin mix antibiotics (Cellgro, Manassas, Va., USA)supplements. Cells were maintained in the log phase at a concentrationof between 1×10⁵ and 1×10⁶ cells/mL.

Friedreich's Ataxia fibroblasts, and control cells ((Coriell, catalognumbers GM04078 and GM08402, respectively). Fibroblasts were cultured in64% (v/v) Eagle's Minimal Essential Medium (MEM), no phenol red withEagle's balanced salt (EBS) and 25% M199 with EBS (GIBCO, Grand island,NY, USA) supplemented with 10% (v/v) Fetal Calf Serum (HyClone, SouthLogan, Utah, USA), 1% penicillin-streptomycin mix antibiotics (Cellgro,Manassas, Va., USA), 10 ng/mL insulin (Sigma, St. Louis, Mo., USA), 10ng/mL basic fibroblast growth factor βFGF (Lonza, Walkersville, Md.,USA) and 2 mM glutamine (HyClone, South Logan, Utah, USA).

CoQ₁₀ deficient lymphocyte and normal lymphocyte cell lines (CoriellCell Repositories, catalog number GM-17932, GM-158151, respectively).CoQ₁₀ deficient lymphocytes were cultured under glucose-free mediasupplemented with galactose for two weeks to force energy productionpredominantly through oxidative phosphorylation rather than glycolysis(Quinzii et al.; Robinson B H, Petrova-Benedict R, Buncic J R, Wallace DC. Nonviability of cells with oxidative defects in galactose medium: Ascreening test for affected patient fibroblast. Biochem. Med. Metab.Biol. 1992; 48:122-126). Lymphocytes were cultured in RPMI 1640 mediumglucose-free (Gibco, Grand Island, N.Y.) supplemented with 25 mMgalactose, 2 mM glutamine and 1% penicillin-streptomycin (Cellgro), and10%, dialyzed fetal bovine serum FBS (<0.5 μg/mL) (GEMINI, Bio-Product).

Leber's cells (Coriell, catalogue number GM10744) were cultured in RPMI(GIBCO, Grand island, NY, USA) with 15% FBS (Fisher Scientific, TX,USA), 2 mM glutamine (HyClone, South Logan, Utah, USA) and 1%penicillin-streptomycin mix antibiotics (Cellgro, Manassas, Va., USA)supplements. Cells were maintained in the log phase at a concentrationof between 1×10⁵ and 1×10⁶ cells/mL.

Alzheimer's fibroblasts (Coriell, catalogue number AG06848) werecultured in Eagle's Minimum Essential Medium with Earle's salts (GIBCO,Grand island, NY, USA) and non-essential amino acids (Invitrogen, N.Y.,USA) with 15% FBS (Fisher Scientific, TX, USA), 2 mM glutamine (HyClone,South Logan, Utah, USA) and 1% penicillin-streptomycin mix antibiotics(Cellgro, Manassas, Va., USA) supplements. Cells were cultured in 75 mlflaks and were maintained at confluency.

Alzheimer's lymphoblasts (Coriell, catalogue number AG06849) werecultured in RPMI (GIBCO, Grand island, NY, USA) with 15% FBS (FisherScientific, TX, USA), 2 mM glutamine (HyClone, South Logan, Utah, USA)and 1% penicillin-streptomycin mix antibiotics (Cellgro, Manassas, Va.,USA) supplements. Cells were maintained in the log phase at aconcentration of between 1×10⁵ and 1×10⁶ cells/mL.

Leigh syndrome cells (Coriell, catalogue number GM13740) were culturedin RPMI (GIBCO, Grand island, NY, USA) with 15% FBS (Fisher Scientific,TX, USA), 2 mM glutamine (HyClone, South Logan, Utah, USA) and 1%penicillin-streptomycin mix antibiotics (Cellgro, Manassas, Va., USA)supplements. Cells were maintained in the log phase at a concentrationof between 1×10⁵ and 1×10⁶ cells/mL.

I. Lipid Peroxidation Assay

Cis-Parinaric Acid Oxidation to Measure Lipid Peroxidation Severalmethods for assaying lipid peroxidation in vitro have been developed(Kuypers et al. (1987) Biochim Biophys Acta. 25, 266-274; Pap et al.(1999) FEBS Lett 453, 278-282; Drummen et al. (2002) Free Radic BiolMed. 33, 473-490). Almost all of these methods are based on inhibitionof free radical-induced oxidation reactions. A widely used fluorescenceassay for lipid peroxidation uses lipid soluble cis-parinaric acid as aprobe. cis-parinaric acid loses its fluorescence (λ_(exc/em): 320/432nm) upon interaction with peroxyl radicals and retains its fluorescencein the presence of radical quenchers. cis-parinaric acid is, however,air sensitive, photolabile and absorbs light in the UV region of thespectrum (at ˜320 nm). However, this region of the spectrum is wheremost compounds have also been found to absorb and emit light. Inpractical terms, the results obtained using cis-parinaric as a probe forlipid peroxidationare confounded due to the overlapping of the compoundsemission spectra with the cis-parinaric emission spectrum.

C₁₁-BODIPY^(581/591) Oxidation to Measure Lipid Peroxidation

To overcome the problem of spectral overlap using cis-parinaric acid, afluorescence assay for lipid peroxidation using a lipophilic probebelonging to the BODIPY class of fluorescent dyes was used.C₁₁-BODIPY^(581/591) (4,4-difluoro-5(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid) fluorescence shifts from red togreen upon oxidation. C₁₁-BODIPY^(581/591) (Molecular Probes, Eugene,Oreg., USA) stock solution concentrations were determined by measuringthe absorption of C₁₁-BODIPY^(581/591) at 582 nm using a molarextinction coefficient of 140,000 mol⁻¹ cm⁻¹ (R. P. Haugland, (1999)Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes,Inc., Eugene, Oreg.). The lipid peroxidation inducer2,2′-Azobis(2-amidino-propane dihydrochloride) (AAPH) and theantioxidant compound α-tocopherol were obtained from Sigma (St. Louis,Mo., USA). Phospholipid bilayers were prepared from1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC) and1,2-dilinoleoyl-phosphatidylcholine (DLPC) and were purchased fromAvanti® polar lipids, INC (Alabaster, Ala., USA).

Preparation of Liposomes

Phosphotidylcholine (PC) liposomes were prepared as described before(Guey-Shuang et al. (1982) Lipids. 17, 403-413). Briefly, DLPC (25 mg)and SOPC (25 mg) were dissolved in chloroform and the solvent wasremoved by nitrogen evaporation (˜2 hours to give a thin film of PC in around bottom flask. The lipid film was hydrated with 50 mL of 10 mMTris-HCl (pH 7.4), 100 mM KCl, shaken and sonicated for 15 seconds. Theliposomes obtained were filtered several times through 0.2 μM membranefilter.

Measurement of C₁₁-BODIPY^(581/591) Oxidation

C₁₁ BODIPY^(581/591) was incorporated into liposomes and oxidized byperoxyl radicals derived from the decomposition of AAPH in presence andabsence of the compounds. Liposomes (1 mg/mL), suspended in 10 mMTris-HCl (pH 7.4), 100 mM KCl, were transferred to a quartz 1 mL cuvetteand placed in a Varian Cary Eclipse fluorometer (Varian, Cary, N.C.)equipped with a thermostatted cuvette holder at 40° C. Liposomes werepre-incubated for 10 min with 200 nM C₁₁ BODIPY^(581/591) to allow theirincorporation into the lipid phase of the liposomes. After the additionof AAPH (10 mM) the decay of red fluorescence was followed atλ_(exc)=570 nm, λ_(em)=600 nm. Relative fluorescence units werenormalized to 100% intensity. Results obtained were verified byrepeating experiments N=3 independent experiments.

Measurement of C₁₁-BODIPY^(581/591) Oxidation in Cell Culture

Lipid peroxidation in cells was detected by utilizing theoxidant-sensitive lipophilic probe C₁₁ BODIPY^(581/591). Briefly, cells(5×10⁵ cell/mL) were treated with the test compounds at finalconcentrations of 1 and 2.5 μM, and incubated at 37° C. for 4 or 24 h ina humidified atmosphere containing 5% CO₂ in air. Cells were treatedwith 500 nM C₁₁ BODIPY^(581/591) in phenol red-free RPMI-1640 media andincubated at 37° C. in the dark for 30 minutes. The cells were washedtwice with phosphate buffered saline and oxidative stress was inducedwith 5 mM DEM in phenol red-free RPMI-1640 media for 90 minutes. Treatedcells were collected by centrifugation at 300×g for 3 minutes and thenwashed twice with phosphate buffered saline. Cells were re-suspended in250 μL of phosphate buffered saline and were analyzed by FACS (FACSCalibur flow cytometer, Becton Dickinson) to monitor the change inintensity of the C₁₁ BODIPY^(581/591) green (oxidized) fluorescencesignal.

Assay for Thiobarbituric Acid Reactive Species (TBARS)

Lipid peroxidation by hydrogen peroxide in bovine heart mitochondrialmembranes was determined by measuring the amount of thiobarbituric acidreactive substances released. Bovine heart mitochondria (1 mg protein)prepared as described by Smith (38) were added to 800 μL of 50 mMphosphate buffer, pH 8.0, and subjected to oxidative stress by theaddition of 25 mM glucose and 1 U/mL glucose oxidase from Aspergillusniger. Samples were incubated with or without test compounds at 37° C.for 30 minutes. Two hundred μL each of 1% (w/v) thiobarbituric acid and35% (v/v) perchloric acid, as well as 0.1% (w/v) butylatedhydroxytoluene (from a 2% stock solution in DMSO) were added. Sampleswere heated at 100° C. for 15 minutes. One-mL aliquots of each samplewere taken and diluted in 2 mL of water, then extracted once with 2 mLof n-butanol. Triplicate 500-μL aliquots were taken from the butanolphase and transferred to a quartz cuvette. TBARS were determinedfluorometrically from the emission spectrum (λ_(ex) 515 nm; λ_(em) 550nm) using a Varian fluorimeter. The malondialdehyde concentration wasdetermined based on a standard curve created using serial dilutions of10 mM 1,1,3,3-tetraethoxypropane hydrolyzed in 1% (v/v) H₂SO₄ at 4° C.overnight. The malondialdehyde concentration was expressed as nmolesmanoldialdehyde per mg protein. Protein in aliquots of the homogenateswas determined by the bicinchoninic acid method.

II. Reactive Oxygen Species (ROS) Assay

Cellular ROS production can be monitored using2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (LeBel et al.(1992) Chem. Res. Toxicol. 5, 227-231) (Molecular Probes, Eugene, Oreg.,USA), a membrane permeable and oxidant-sensitive fluorescent dye.DCFH-DA is a non-fluorescent derivative of fluorescein that emitsfluorescence after being oxidized by hydrogen peroxide and other ROS.The emitted fluorescence is directly proportional to the concentrationof hydrogen peroxide. DCFH-DA is nonionic and nonpolar and is easilytaken up by cells. Once inside the cell, DCFH-DA is hydrolyzed bycellular esterases to non-fluorescent DCFH which traps the dye in thecell. In the presence of ROS including hydrogen peroxide, DCFH isoxidized to the highly fluorescent compound dichlorofluorescein (DCF).The intracellular DCF fluorescence is used as an index of cellular ROSproduction.

Cellular oxidative stress was induced by pharmacological depletion ofglutathione (GSH) using the chemical diethylmaleate (DEM). Cells (1×10⁶cell/mL) were plated (1 mL) in twelve well plate and treated with theinvention compounds (final concentration 1, 2.5, 5 or 10 μM), andincubated for fifteen hours at 37° C., 5% CO₂. The compounds tested wereprepared by first making stock solutions (1 mM) in dimethylsulfoxide(DMSO). Cells were treated with 5 mM DEM for 30 minutes and collected bycentrifugation at 300×g for 3 min and then washed twice with PhosphateBuffer Saline (PBS) (Invitrogen, N.Y., USA). Cells were re-suspended inPBS buffer+10 mM glucose and incubated at 37° C. in the dark for 20 minwith 10 μM DCFH-DA. Cells were collected by centrifugation at 300×g for3 min and then washed twice with PBS buffer. The samples were analyzedimmediately by flow cytometry (Becton-Dickinson FACS Caliber), (CellQuest software, BD Biosciences) using 488 nm excitation laser and FL1-Hchannel 538 nm emission filter. In each analysis, 10,000 events wererecorded after cell debris were electronically gated out. Resultsobtained were verified by running duplicates and repeating experimentsN=3 independent experiments. Authentic hydrogen peroxide was used as apositive control.

III. Superoxide (O₂ ^(−.)) Assay

Cellular Superoxide production can be monitored using Dihydroethidium(DHE), a fluorogenic probe that is highly selective for Superoxide amongROS (Bindokas V P, J Neurosci 16: 1324-1336, 1996) (Invitrogen, USA).The emitted fluorescence is directly proportional to the concentrationof superoxide radical. It is cell-permeable and reacts with superoxideanion to form ethidium, which in turn intercalates in thedeoxyribonucleic acid, thereby exhibiting a red fluorescence (Hwan H Y,Oncology Reports 21: 253-261, 2009). Cellular Superoxide generation wasinduced by pharmacological inhibition of the mitochondrial electrontransport between cytochrome b and c, using Antimycin A (Alexander A.Biochim Biophys Acta 767:120-129, 1984) (Sigma-Aldrich).

Cells (1×10⁶ cell/mL) were plated (1 mL) in twenty four well plate andtreated with the invention compounds (final concentration 1, 2.5, 5 or10 μM), and incubated for fifteen hours at 37° C., 5% CO₂. The compoundstested were prepared by first making stock solutions (1 mM) indimethylsulfoxide (DMSO). Cells were treated with 50 μM Antimycin A fortwo hours and then, with 60 μM DHE. The samples were analyzedimmediately by flow cytometry (Becton-Dickinson FACS Caliber), (CellQuest software, BD Biosciences) using 488 nm excitation laser and FL2-Hchannel 585 nm emission filter. In each analysis, 10,000 events wererecorded after cell debris were electronically gated out. Resultsobtained were verified by running duplicates and repeating experimentsN=3 independent experiments. Mn(III)tetrakis(4-benzoic acid)porphyrinChloride (MnTBAP) (Sigma-Aldrich, Mo., USA) was used as a positivecontrol.

IV. Mitochondrial Membrane Potential (Δψ_(n)) Assay

Measurement of Mitochondrial Membrane Potential (Δω_(m))(FACS). For thedetermination of Δψ_(m), cells were pre-treated with or without the testcompounds. The cells were treated with 5 mM DEM for 120 minutes,collected by centrifugation at 300×g for 3 minutes and then washed twicewith phosphate buffered saline. The cells were re-suspended in PBSbuffer and incubated at 37° C. in the dark for 15 minutes with 250 nMTMRM (a cationic dye which accumulates within mitochondria in accordancewith the Δ_(ψm)Nernst potential). Cells were collected by centrifugationat 300×g for 3 minutes and then washed twice with phosphate bufferedsaline. The samples were analyzed immediately by flow cytometry using488 nm excitation laser and the FL2-H channel. The results obtained wereverified in three independent experiments. The protonophore FCCP (30 μM)was used to dissipate the chemiosmotic proton gradient (ΔμH⁺) and servedas a control for loss of Δω_(m). In each analysis, 10,000 events wererecorded.

V. Trypan Blue Cell Viability Assay

Cell viability determined by trypan blue exclusion assay: This techniquewas used to assess the cytoprotective effects of the invention compoundsin cultured cells pharmacologically treated to induce cell death by GSHdepletion. DEM was used to deplete cellular GSH and induce oxidativestress. The viability of DEM-treated cells was determined by theirability to exclude the dye trypan blue. Viable cells exclude trypanblue; whereas, non-viable cells take up the dye and stain blue. Briefly,cells were seeded at a density of 1×10⁶ cells/mL and treated withdifferent concentrations of the invention compounds. Cells wereincubated at 37° C. in a humidified atmosphere of 5% CO₂ in air forthree hours with 5 mM DEM. Cell viability was determined by stainingcells with 0.4% trypan blue using a hemocytometer. At least 500 cellswere counted for each experimental group. At the time of assay, >95% ofDEM-treated cells were trypan blue positive; whereas, in non-DEM treatedcontrol cell cultures >95% cells were viable. Cell viability wasexpressed as the percentage of control. Data are expressed asmeans±S.E.M (n=3).

VI. Cell Viability Assays (FACS)

Cell viability and cytotoxicity were determined by simultaneous staininglive and dead cells using a two-color fluorescence assay, the Live/Dead®Viability/Cytotoxicity Kit (Molecular Probes). This assay is used tomeasure two recognized parameters of cell viability, intracellularesterase activity and plasma integrity. The membrane-impermeant DNA dyeethidium homodimer-1 (EthD-1) was used to identify dead cells whoseplasma membrane integrity was disrupted. The membrane-permeant dyecalcein-AM was used to label live cells. It penetrates into the cellswhere it is metabolized by cytoplasmic esterases and becomes afluorescent but membrane-impermeant probe which is retained in viablecells. Cells were incubated overnight in RPMI medium (control) and inthe presence of test compound and then treated with DEM for 3 to 6hours. Cells were stained with 0.2 μM calcein-AM and 0.4 μM EthD-1.After 15 minutes, flow cytometry analysis was carried out usingexcitation at 488 nm. The green-fluorescent (539 nm) FL1-H channel,live-cell population appears in the lower right quadrant and thered-fluorescent (585 nm) FL2-H channel dead-cell population appears inthe upper left quadrant. In each analysis, 10,000 events were recorded.Results obtained were verified in three independent experiments.

VII. Calcein-AM Cell Viability Assay

A cell viability assay using the dye calcein acetoxymethyl (AM) was usedto determine the effects of invention compounds on GSH-mediated celldeath in primary FRDA patient derived fibroblasts (Jauslin et al. (2002)Human Molecular Genetics. 11, 3055-3063) FRDA fibroblasts were treatedwith L-buthionine (S,R)-sulfoximine (BSO) to inhibit de novo synthesisof GSH (Griffith et al. (1979) J. Biol. Chem. 254, 7558-7560) causingoxidative stress, plasma membrane damage and cell death. Fibroblastsfrom Friedreich Ataxia patients, but not control cells, die after GSHdepletion on incubation with BSO (Jauslin et al. (2002) Human MolecularGenetics. 11, 3055-3063). Cell viability was determined using calcein-AM(Molecular Probe, Eugene, Oreg.). In live cells, non-fluorescent calceinAM is hydrolyzed by intracellular esterases to produce the stronglygreen fluorescent anion calcein.

BSO (L-buthionine (S,R)-sulfoximine) and (+)-alpha-tocopherol werepurchased from Sigma Chemicals and calcein AM was purchased fromMolecular Probes. Cell were grown in 75 cm² culture flasks (T75) andincubated at 37° C. in a humidified atmosphere of 5% CO₂ in air. Cellswere fed twice a week and split every third day at a ratio of 1:3 uponreaching confluency. The invention compounds and reduced and oxidizedforms of idebenone and alpha-tocopherol were reconstituted in DMSO orethanol to provide 2.5 mM stock solutions.

The compounds were screened according to the previous protocol (Jauslinet al. (2002) Human Molecular Genetics. 11, 3055-3063): Fibroblasts wereseeded in 96 well microtiter black-walled cell culture plates (Costar,Corning, N.Y., USA) at a density of 3000 cells per well (100 μL). Theplates were incubated overnight at 37° C. in a humidified atmosphere of5% CO₂ in air to allow attachment of the cells to the culture plate.Serial dilutions of intervention and references compounds were made fromtheir respective stock solutions to give a total volume of 150 μL ineach well. Plates were incubated overnight in cell culture. Thefollowing day, 50 μL of a 4 mM BSO solution (in culture media) was addedto each well to provide a final BSO concentration of 1 mM. Cellviability was assessed after first signs of toxicity appeared inBSO-treated cells (typically after 24-30 hours) by examining culturesunder phase-contrast microscopy. The cell culture medium was discardedby aspiration and each well of the cell culture plate washed withpre-warmed HSSB to remove serum esterase activity. Cells were thentreated in with 200 μL of 1.2 μM calcein-AM in HSSB for 60 min at 37° C.in the dark to allow the dye to enter the cell and be cleaved byesterases. The negative control/background was 200 μL of HSSB buffer.Fluorescence intensities were measured with a Spectramax M5spectrofluorometer (Molecular Devices, Sunnyvale, Calif., USA) usingexcitation and emission of 485 nm and 525 nm respectively. Theintervention compounds were assayed in triplicate. The solvent vehiclesused either DMSO or ethanol did not affect cell viability at theconcentrations (0.5-1%) used in the assay. The viability of non-BSOtreated fibroblasts was set as 100%, and the viability of theBSO-treated and sample-treated cells was calculated relative to thisvalue. Cell viability was expressed as the percentage of control. Dataare expressed as means±S.E.M (n=3).

VIII. Cytochrome c Reduction Assay

The rate of cytochrome c (10 μM) reduction was measured by monitoringthe change in absorbance at 550 nm. Briefly the reaction was initiatedby addition of 100 μM of the invention compounds to a mixture containing50 mM phosphate buffer, 0.1 mM EDTA, pH 7.8, and 10 μM cytochrome c(Sigma, St. Louis, Mo. USA). For cytochrome c reduction by superoxide,xanthine oxidase (0.01 IU/mL) (Sigma, St. Louis, Mo. USA) was used inpresence of xanthine (50 μM).

IX. Total Intercellular ATP Concentration Assay

The reductions of mitochondrial respiratory chain activity in CoQ₁₀deficient patients have been reported (Quinzii G M, Lopez L C,Von-Moltke J, Naini A, Krishna S, Schuelke M, Salviati L, Navas P,DiMauro S, and Hirano, M. Respiratory chain dysfunction and oxidativestress correlate with severity of primary CoQ10 deficiency. FASEB J.2008; 22:1874-1885). The use of CoQ₁₀ analogues to normalize and restorethe respiratory chain activities could provide valuable therapeuticapproach. We have evaluated the efficiency of oxidative phosphorylationin CoQ₁₀ deficient lymphocyte (GM17932) in presence of tested CoQ₁₀analogues by measuring total cellular ATP content using (ViaLight® PlusATP monitoring reagent kit, Lonza).

Briefly, lymphocytes (2×10⁵ cell/mL), were plated (1 mL in 12-wellplates) and treated with the test compounds at final concentrations of5, 10 μM, and 25 μM and incubated at 37° C. for 48 h in a humidifiedatmosphere containing 5% CO₂ in air. The test compounds were prepared byfirst making 20 mM stock solutions in DMSO. Cells were transferred (100μL) to 96-well microtiter black-walled cell culture plates (Costar,Corning, N.Y.). The total intracellular ATP level was measured in aluminator (Clarity™ luminescence microplate reader) with the ATPBioluminescence Assay Kit (ViaLight® Plus ATP monitoring reagent kit,Lonza) following the manufacturer's instructions. The standard curve ofATP was obtained by serial dilution of 1 mM ATP solution. Aftercalibration against the ATP standard, the ATP content of the cellextract was determined and normalized for protein content in the cell.FIG. 1 shows that the cellular content of ATP are significantly lower inCoQ₁₀ deficient than normal lymphocyte.

X. NADH Oxidase Inhibition Assay

Beef heart mitochondria were obtained by a large-scale procedure.Inverted submitochondrial particles (SMP) were prepared by the method ofMatsuno-Yagi and Hatefi (J. Biol. Chem. 260 (1985), p. 14424), andstored in a buffer containing 0.25 M sucrose and 10 mM Tris-HCl (pH 7.4)at −80° C. Inhibitory effects of compounds on bovine heart mitochondrialcomplex (I, III, IV) were evaluated. Maximal dimethyl sulfoxideconcentration never exceeded 2% and had no influence on the controlenzymatic activity. Beef heart SMP were diluted to 0.5 mg/mL. Theenzymatic activities were assayed at 30° C. and monitoredspectrophotometrically with a Beckman Coulter DU-530 (340 nm, ε=6.22mM⁻¹ cm⁻¹). NADH oxidase activity was determined in a reaction medium(2.5 mL) containing 50 mM Hepes, pH 7.5, containing 5 mM MgCl₂. Thefinal mitochondrial protein concentration was 30 μg/mL. After thepre-equilibration of SMP with inhibitor for 5 min, the initial rateswere calculated from the linear portion of the traces.

XI. Mitochondrial Bioenergetics Assessment

The use of CoQ₁₀ analogues and methylene blue analogues to normalize andrestore the respiratory chain activities provides valuable therapeuticapproach for mitochondrial diseases. The reductions of mitochondrialrespiratory chain activity in Friedreich ataxia (FRDA) patients havebeen reported. The efficiency of oxidative phosphorylation was evaluatedin FRDA lymphocyte (GM15850) in presence of tested CoQ₁₀ analogues andmethylene blue analogues by measuring total cellular ATP content using(ViaLight®-Plus ATP monitoring reagent kit, Lonza).

FRDA lymphocyte cell lines were obtained from Coriell Cell Repositories.FRDA lymphocytes were cultured under glucose-free media supplementedwith galactose for two weeks to force energy production predominantlythrough oxidative phosphorylation rather than glycolysis. Lymphocyteswere cultured in RPMI 1640 medium glucose-free supplemented with 25 mMgalactose, 2 mM glutamine and 1% penicillin-streptomycin, and 10%,dialyzed fetal bovine serum FBS (<0.5 μg/mL). Briefly, lymphocytes(2×10⁵ cell/mL), were plated (1 mL in 12-well plates) and treated withthe test compounds at final concentrations of 50, 125. 250, 1000, and5000 nM, and incubated at 37° C. for 48 h in a humidified atmospherecontaining 5% CO₂ in air. Cells were transferred (100 μL) to 96-wellmicrotiter black-walled cell culture plates. The total intracellular ATPlevel was measured in a luminator (Clarity™ luminescence microplatereader) with the ATP Bioluminescence Assay Kit (ViaLight®-Plus ATPmonitoring reagent kit, Lonza) following the manufacturer'sinstructions. Carbonyl cyanide-p-trifluormethoxy-phenylhydrazone (FCCP)and oligomycin were used as control for inhibition of ATP synthesis.

Results

The compounds of the disclosure were assayed for NADH oxidase inhibitionactivity. The results, expressed as % activity, are shown in Table 1,below.

TABLE 1 NADH oxidase (complex I, III, IV) activity (%) 10 μM 5 μM l μMCPD-2 11.9 ± 1.6  12 ± 0.8 20.1 ± 1.4 CPD-3 70.4 ± 5.5 76.7 ± 6.9  92.9± 10.9 CPD-5 33.5 ± 2.2 44.5 ± 3.6 74.5 ± 6.9 CPD-4 10.7 ± 1.3 12.7 ±1.7 17.1 ± 1  CPD-1 13.2 ± 1  20.7 ± 2.2 29.7 ± 3.8

The compounds of the disclosure were assayed for suppression of ReactiveOxygen Species (ROS) production in cultured CEM leukemia cellspretreated with DEM. The results, shown in Table 2, are expressed as %of scavenging activity. “Untreated control” represents no DEM treatment,while the “treated control” represents DEM treatment alone.

TABLE 2 Compounds 10 μM 2.5 μM 0.25 μM Untreated Control 100  100  100 Treated Control 0 0 0 CPD-3 67.0 ± 5.9 42.2 ± 2.9  4.6 ± 5.4 CPD-5  0 ±10 26.4 ± 4.4 21.0 ± 5.5 CPD-4 91.0 ± 1.5 81.8 ± 3.7  41.4 ± 14.9 CPD-120.0 ± 4.4 22.2 ± 3.5 23.4 ± 1.6 CPD-2 35.2 ± 3.9 25.3 ± 5.2 31.1 ± 1.3

Cell viability after the treatment with the compounds of the disclosurewas assayed using an FRDA cell line and a trypan blue exclusion assay(as described above). The results are expressed as % viability, andshown in Table 3. “Untreated” means control cells grown without anystress challenge; “Treated” means cells stressed with DEM, but nottreated with the test compound.

TABLE 3 Compound 2.5 μM 0.5 μM 0.1 μM Untreated Treated CPD-1 30 ± 6 42± 3 25 ± 6 93 ± 7 21 ± 8 CPD-3 25 ± 5 19 ± 3 19 ± 6 96 ± 2 12 ± 3 CPD-514 ± 3 17 ± 7 12 ± 6 96 ± 2 12 ± 3 CPD-4 35 ± 9 21 ± 9 17 ± 5 96 ± 2 12± 3 CPD-2 15 ± 4 14 ± 5 10 ± 3 96 ± 2 12 ± 3 0.1 μM 0.5 μM 2.5 μMUntreated Treated CPD-6 27 ± 7  30 ± 13 24 ± 6 93 ± 7 21 ± 8 CPD-9  31 ±12 28 ± 7 32 ± 5 97 ± 5 18 ± 3 CPD-12 15 ± 6 29 ± 3 39 ± 3 97 ± 5 18 ± 3CPD-7 36 ± 6 14 ± 5 20 ± 3 97 ± 9 17 ± 2 CPD-10 19 ± 6  20 ± 13 32 ± 897 ± 9 17 ± 2 CPD-13 17 ± 3 14 ± 2 19 ± 5 97 ± 9 17 ± 2 CPD-8 19 ± 6 15± 5 18 ± 2 97 ± 9 17 ± 2 CPD-11 12 ± 5  6 ± 3  8 ± 3 96 ± 2 12 ± 3

The compounds of the disclosure were also assayed for the lipidperoxidation suppression. The results of the suppression of lipidperoxidation in cultured leukemia CEM lymphocytes treated with diethylmaleate (DEM) is shown in Tables 4 and 5 below. Values have beencalculated as (100−% mean)×100/(% mean of the treated control).“Untreated control” represents no DEM treatment, while the “treatedcontrol” represents DEM treatment

TABLE 4 Scavenging activity (%) Compound 1 μM 2.5 μM 5 μM untreatedcontrol 100  100  100  treated control 0 0 0 CPD-3 14 ± 4.1 20 ± 5.4 9.6± 2.8 CPD-5 11 ± 1.8 16 ± 1.8 2.2 ± 2.3 CPD-4 18 ± 2.2 52 ± 1.5  94 ±0.2 CPD-1 0 0 0 CPD-2  7 ± 2.8 13 ± 4.2 0

TABLE 5 Scavenging activity (%) Compound 1 μM 5 μM untreated control 100100 treated control 0 0 CPD-6 0 0 CPD-9 0 0 CPD-12 0 0 CPD-7 0 0 CPD-100 0 CPD-13 0 0 CPD-8 10 ± 4.3 20 ± 3.4 CPD-11  7 ± 2.3 15 ± 3.3

In addition, effect of the compounds of the invention on lipidperoxidation induced by oxygen radicals is illustrated in FIGS. 1 and 2.Oxygen radicals were generated from thermal decomposition of AAPH (10mM) in phospholipids liposomes in Tris-HCl buffer containingC₁₁-BODIPY^(581/591) (200 nM) at 40° C. The red fluorescence decay ofthe probe was monitored (λ_(ex)=570, λ_(em)=600 nm) over 30 minutes.

The efficiency of oxidative phosphorylation was evaluated in FRDAlymphocyte in presence of methylene blue and the compounds of thedisclosure. After forty hours, methylene blue and the compound of thedisclosure (CPD-18) significantly increased cellular ATP levels in FRDAlymphocyte at lower concentration (FIG. 3). CPD-18 was superior tomethylene blue in maintaining ATP levels when used at higherconcentrations.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be incorporated within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated herein by referencefor all purposes.

We claim:
 1. A compound of formula:

wherein R³ is hydrogen, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, or—OR⁷, each optionally substituted with one to four substituents selectedfrom halogen, —CN, —NO₂, C₁-C₆ alkyl, halo(C₁-C₆ alkyl), —OR⁸, —NR⁸ ₂,—CO₂R⁸, —CONR⁸ ₂, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, aryl,heteroaryl, and heterocyclyl, wherein each cycloalkyl, cycloalkenyl,aryl, heteroaryl, and heterocyclyl are optionally substituted with R⁹;where each R⁷ independently is hydrogen, C₁-C₆ alkyl, or halo(C₁-C₆alkyl); where each R⁸ independently is hydrogen, C₁-C₆ alkyl, halo(C₁-C₆alkyl), C₃-C₈ cycloalkyl, aryl, heteroaryl, heterocyclyl, aryl(C₁-C₆alkyl), C₃-C₈ cycloalkyl(C₁-C₆ alkyl), aryl(C₁-C₆ alkyl),heteroaryl(C₁-C₆ alkyl), or heterocycle(C₁-C₆ alkyl), wherein eachcycloalkyl, aryl, heteroaryl, and heterocyclyl are optionallysubstituted with R⁹; where each R⁹ independently is halogen, —CN, —NO₂,—N₃, C₁-C₆ alkyl, halo(C₁-C₆ alkyl), C₁-C₆ alkoxy, amino,C₁-C₆alkylamino, or diC₁-C₆alkylamino; R⁴ and R⁵ are independentlyC₄-C₂₀ alkyl, C₄-C₂₀ alkenyl, or C₄-C₂₀ alkynyl, each optionallysubstituted with one to four substituents selected from halogen, —CN,—NO₂, C₁-C₆ alkyl, halo(C₁-C₆ alkyl), —OR⁸, —NR⁸ ₂, —CO₂R⁸, —CONR⁸ ₂,C₃-C₈ cycloalkyl, C₃-C₈ cycloalkenyl, aryl, heteroaryl, and heterocycle,wherein each cycloalkyl, cycloalkenyl, aryl, heteroaryl, andheterocyclyl are optionally substituted with R⁹; each R⁶ is hydrogen,C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, C₃-C₈ cycloalkyl, C₃-C₈cycloalkenyl, aryl, heteroaryl, heterocycle, or —OR¹⁰, wherein eachalkyl, alkenyl, and alkynyl are optionally substituted with one to foursubstituents selected from halogen, —CN, —NO₂, C₁-C₆ alkyl, halo(C₁-C₆alkyl), —OR⁸, —NR⁸ ₂, —CO₂R⁸, —CONR⁸ ₂, C₃-C₈ cycloalkyl optionallysubstituted with R⁹, C₃-C₈ cycloalkenyl optionally substituted with R⁹,aryl optionally substituted with R⁹, heteroaryl optionally substitutedwith R⁹, and heterocyclyl optionally substituted with R⁹; where R¹⁰ isC₁-C₆ alkyl, halo(C₁-C₆ alkyl), C₃-C₈ cycloalkyl, aryl, heteroaryl,heterocycle, aryl(C₁-C₆ alkyl), C₃-C₈cycloalkyl(C₁-C₆ alkyl), aryl(C₁-C₆alkyl), heteroaryl(C₁-C₆ alkyl), or heterocycle(C₁-C₆ alkyl), whereineach cycloalkyl, aryl, heteroaryl, and heterocycles are optionallysubstituted with R⁹.
 2. The compound according to claim 1, wherein R³ ishydrogen, optionally substituted C₁-C₂₀ alkyl, or —OR⁷.
 3. The compoundaccording to claim 2, wherein R³ is hydrogen.
 4. The compound accordingto claim 2, wherein R³ is optionally substituted C₁-C₂₀ alkyl.
 5. Thecompound according to claim 1, wherein R³ is C₁-C₂₀ alkyl optionallysubstituted with —OR⁸, C₃-C₈ cycloalkyl, C₃-C₈cycloalkenyl, or aryl,wherein each cycloalkyl, cycloalkenyl, and aryl are optionallysubstituted with R⁹.
 6. The compound according to claim 5, wherein R³ isC₁-C₂₀ alkyl optionally substituted with C₃-C₈ cycloalkyl, C₃-C₈cycloalkenyl, or aryl, wherein each cycloalkyl, cycloalkenyl, and arylare optionally substituted with R⁹.
 7. The compound according to claim1, wherein R³ is —OR⁷ and R⁷ is C₁-C₆ alkyl.
 8. The compound accordingto claim 1, wherein R⁴ is optionally substituted C₄-C₂₀ alkyl.
 9. Thecompound according to claim 8, wherein R⁴ is C₄-C₂₀ alkyl.
 10. Thecompound according to claim 1, wherein R⁵ is optionally substitutedC₄-C₂₀ alkyl.
 11. The compound according to claim 10, wherein R⁵ isC₄-C₂₀ alkyl.
 12. The compound according to claim 1, wherein R⁶ ishydrogen or optionally substituted C₁-C₂₀ alkyl.
 13. The compoundaccording to claim 12, wherein R⁶ is hydrogen.
 14. The compoundaccording to claim 12, wherein R⁶ is C₁-C₂₀ alkyl optionally substitutedwith —OR⁸, C₃-C₈ cycloalkyl, C₃-C₈cycloalkenyl, or aryl, wherein eachcycloalkyl, cycloalkenyl, and aryl are optionally substituted with R⁹.15. The compound according to claim 14, wherein R⁶ is C₁-C₂₀ alkyloptionally substituted with C₃-C₈ cycloalkyl, C₃-C₈cycloalkenyl, oraryl, wherein each cycloalkyl, cycloalkenyl, and aryl are optionallysubstituted with R⁹.
 16. A pharmaceutical composition comprising acompound or a pharmaceutically acceptable salt according to claim 1 andan acceptable carrier, excipient and/or diluent.
 17. A method oftreating or suppressing diseases associated with decreased mitochondrialfunction resulting in diminished ATP production and/or oxidative stressand/or lipid peroxidation, comprising administering an effective amountof a compound or a pharmaceutically acceptable salt according to claim1, wherein the disease is selected from the group consisting ofFriedreich's ataxia, Leber's Hereditary Optic Neuropathy, Kearns-SayreSyndrome, Mitochondrial Encephalomyopathy with Lactic Acidosis andStroke-Like Episodes, Leigh syndrome, amyotrophic lateral sclerosis,Huntington's disease, Alzheimer's disease and Parkinson's disease.
 18. Amethod according to claim 17, wherein the disease is Friedreich'sataxia.
 19. The compound of claim 1, which is:

7-(didecylamino)-3H-phenothiazine-3-one; or

7-(dipentylamino)-3H-phenothiazin-3-one.